U.S. patent application number 10/187498 was filed with the patent office on 2003-11-06 for methods for the production of purified recombinant human uteroglobin for the treatment of inflammatory and fibrotic conditions.
Invention is credited to Pilon, Aprile L., Welch, Richard W..
Application Number | 20030207795 10/187498 |
Document ID | / |
Family ID | 29273231 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207795 |
Kind Code |
A1 |
Pilon, Aprile L. ; et
al. |
November 6, 2003 |
Methods for the production of purified recombinant human
uteroglobin for the treatment of inflammatory and fibrotic
conditions
Abstract
The present invention relates generally to the production of
recombinant human uteroglobin (rhUG) for use as a therapeutic in
the treatment of inflammation and fibrotic diseases. More
particularly, the invention provides processes, including broadly
the steps of bacterial expression and protein purification, for the
scaled-up production of rhUG according to current Good
Manufacturing Practices (cGMP). The invention further provides
analytical assays for evaluating the relative strength of in vivo
biological activity of rhUG produced via the scaled-up cGMP
processes.
Inventors: |
Pilon, Aprile L.;
(Gaithersburg, MD) ; Welch, Richard W.;
(Gaithersburg, MD) |
Correspondence
Address: |
Barry Evans, Esq.
Kramer Levin Naftalis & Frankel LLP
919 THIRD AVENUE
New York
NY
10022
US
|
Family ID: |
29273231 |
Appl. No.: |
10/187498 |
Filed: |
July 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10187498 |
Jul 2, 2002 |
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09898616 |
Jul 2, 2001 |
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09898616 |
Jul 2, 2001 |
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08864357 |
May 28, 1997 |
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6255281 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/472; 435/69.6; 435/7.32; 514/12.2; 536/23.5 |
Current CPC
Class: |
C07K 14/4721 20130101;
A61K 38/1709 20130101 |
Class at
Publication: |
514/6 ; 435/69.6;
435/252.3; 435/472; 536/23.5; 435/7.32 |
International
Class: |
A61K 038/41; G01N
033/554; G01N 033/569; C07H 021/04; C12P 021/04; C12N 015/74; C12N
001/21 |
Claims
What is claimed is:
1. A bacterial expression system for the production of rhUG
comprising a synthetic gene which codes for human UG, wherein the
synthetic gene comprises Seq. ID. Nos. 1-4.
2. The expression system of claim 1, wherein the synthetic gene
further comprises Met-Ala-Ala at the N terminus of the synthetic
gene.
3. A bacterial expression system for production of rhUG comprising
a human cDNA sequence which codes for human UG wherein the gene
further comprises Met-Ala-Ala at the N-terminus of the
sequence.
4. The expression system of claim 3, wherein the expression system
further comprises an approximately 2.8 kb par sequence.
5. A method of producing a rhUG research seed bank comprising: a.
inoculating onto a growth medium at least one colony of a bacterial
strain comprising a rhUG expression system; b. incubating the
inoculated growth medium until a stationary phase is reached; c.
adding glycerol to the inoculated growth medium; d. freezing the
culture in aliquot portions; and e. storing the frozen aliquot
portions at a temperature below about -50 C.
6. The method of claim 5, wherein the inoculated growth medium is
incubated until an optical density measured between 550 nm to 660
nm of about 0.8 AU to 1.5 AU is reached.
7. The method of claim 5, wherein the cryopreservative comprises
glycerol.
8. The method of claim 5, wherein the aliquot portion is about 1
ml.
9. The method of claim 5, wherein the storage temperature is
between about -70 and about -90.degree. C.
10. A method of producing a rhUG master cell bank comprising: a.
inoculating a suitable incubating broth with an aliquot portion of
a rhUG research seed bank to form a bacterial culture; b.
incubating the bacterial culture; c. adding a cryopreservative to
the bacterial culture to form a cryopreserved solution; d.
transferring a portion of the cryopreserved solution to a cryovial;
and e. storing the cryovial at a temperature below about -60 C.
11. The method of claim 10, wherein the culture is incubated until
an optical density measured between 550 nm to 660 nm of about 0.8
AU to 1.5 AU reached.
12. The method of claim 10, wherein the cryopreservative comprises
glycerol.
13. The method of claim 10, wherein the portion transferred to a
cryovial is about 1 ml.
14. The method of claim 10, wherein the storage temperature is
between about -70 and about -90 C.
15. A method of producing a rhUG production cell bank comprising:
a. inoculating a suitable incubating broth with an aliquot portion
of a rhUG master cell bank to form a bacterial culture; b.
incubating the bacterial culture; c. adding a cryopreservative to
the bacterial culture to form a cryopreserved solution; d.
transferring a portion of the cryopreserved solution to a cryovial;
and e. storing the cryovial at a temperature below about -60 C.
16. The method of claim 15, wherein the bacterial culture is
incubated until an optical density measured between 550 nm to 660
nm of about 0.8 AU to 1.5 AU is reached.
17. The method of claim 15, wherein the cryopreservative comprises
glycerol.
18. The method of claim 15, wherein the portion transferred to a
cryovial is about 1 ml.
19. The method of claim 15, wherein the storage temperature is
between about -70 and about -90 C.
20. A method of expressing rhUG comprising the steps of: a.
providing a production seed cell bank culture comprising an
expression vector capable of expressing rhUG; b. inoculating a
broth medium with the production seed cell bank culture to form an
inoculum; c. incubating the bacterial culture formed in step b; d.
inoculating a large scale fermenter with the inoculum formed in
step c to form a fermentation culture; e. incubating the
fermentation culture within the large scale fermenter; f. adding an
induction agent to the fermentation culture to induce the
expression of rhUG; and g. harvesting the fermentation culture
after step f.
21. The method of claim 20, wherein the expression vector comprises
Seq. ID Nos. 1-4.
22. The method of claim 20, wherein the inoculum is incubated for a
period between about 12 hours and about 24 hours at a temperature
between about 28.degree. C. and about 36.degree. C.
23. The method of claim 20, wherein the large scale fermenter has
at least a 300 liter capacity.
24. The method of claim 20, wherein the incubation of step e is
continued until an optical density 550 nm to 660 nm until a minimum
OD of 2.0 AU is reached.
25. The method of claim 20, wherein the induction agent comprises
isopropyl-beta-D-thiogalactopyranoside (IPTG).
26. The method of claim 20, wherein of about 1 to about 4 hours
elapses between step f and step g.
27. The method of claim 20, wherein harvesting the fermentation
culture comprises centrifugation.
28. A method of expressing rhUG comprising the steps of: a.
inoculating a large scale fermenter with an inoculum comprising an
expression vector capable of expressing rhUG to form a fermentation
culture; b. incubating the fermentation culture within the large
scale fermenter; c. adding an induction agent to the fermentation
culture to induce the expression of rhUG; and d. harvesting the
fermentation culture.
29. The method of claim 28, wherein the expression vector comprises
Seq. ID Nos. 1-4.
30. The method of claim 28, wherein the large scale fermenter has
at least a 300 liter capacity.
31. The method of claim 28, wherein the incubation of step b is
continued until an optical density 550 nm to 660 nm until a minimum
OD of 2.0 AU is reached.
32. The method of claim 28, wherein the induction agent comprises
isopropyl-beta-D-thiogalactopyranoside (IPTG).
33. The method of claim 28, wherein of about 1 to about 4 hours
elapses between step c and step d.
34. The method of claim 28, wherein harvesting the fermentation
culture comprises centrifugation.
35. A method of purifying rhUG comprising the steps of: a.
providing a bacterial cell paste comprising bacterial cells capable
of overexpressing rhUG; b. lysing the bacterial cell paste to form
a supernatant; c. filtering the supernatant formed in step b
through a first nominal molecular weight cut off (NMWCO) membrane
to form a first permeate; d. concentrating the first permeate
formed in step c by the use of a second NMWCO membrane; e. loading
the concentrated permeate formed in step d onto an anion exchange
column to form a first eluate; f. concentrating the first eluate
formed in step e by the use of a third NMWCO membrane to form a
second concentrate; g. loading the second concentrate formed in
step f onto a Hydroxyapatite (HA) column to form a second eluate;
h. separating host-derived proteins from the rhUG in the second
eluate formed in step g to provide purified rhUG; and i. recovering
the purified rhUG formed in step h.
36. The method of claim 35, wherein the synthetic gene expressed in
the bacterial cells comprises Seq. ID Nos. 1-4.
37. The method of claim 35, wherein lysing comprises shearing.
38. The method of claim 35, wherein between step b and step c, cell
debris is removed by centrifugation.
39. The method of claim 35, wherein the membrane of step b is about
a 30K to 100K NMWCO membrane.
40. The method of claim 39, wherein the filtering of step c
comprises the use of a tangential flow filtration (TFF) system.
41. The method of claim 35, wherein the membrane of step d is about
a 5K NMWCO membrane.
42. The method of claim 35, further comprising a cation exchange
chromatography step.
43. The method of claim 42, wherein the cation exchange
chromatography step is substituted for the hydroxyapatite
chromatography step.
44. The method of claim 42, wherein a SP Sepharose Fast Flow
(SPSFF) cation exchange column is used.
45. The method of claim 35, further comprising a hydrophobic
interaction chromatography step.
46. The method of claim 45, wherein the hydrophobic interaction
chromatography step is substituted for another chromatography
step.
47. The method of claim 46, wherein a Phenyl Sepharose Fast
Flow/High Substitution (PSFFHS) column is used.
48. The method of claim 41, wherein the anion exchange column of
step e is a Macro Q anion exchange column.
49. The method of claim 41, wherein the host-derived proteins of
step h are separated with a Chelating Sepharose Fast Flow (CSFF)
resin column.
50. The method of claim 49, wherein the CSFF resin column comprises
copper.
51. The method of claim 50, wherein after step h a positively
charged membrane is placed downstream of the CSFF column forming a
pass through substantially free of host derived proteins.
52. The method of claim 51, wherein the positively charged membrane
is a Sartobind Q TFF membrane.
53. The method of claim 35, wherein the second eluate is
diafiltered through about a 30K NMWCO membrane.
54. The method of claim 35, wherein the rhUG recovered in step i is
substantially free of aggregates.
55. A method of purifying rhUG comprising the steps of: a.
providing bacterial cells capable of overexpressing rhUG; b. lysing
the bacterial cells to form a supernatant liquid; c. filtering the
liquid through a molecular weight cut off (NMWCO) membrane; d.
loading the liquid onto an exchange column; e. separating
host-derived proteins from the rhUG to provide purified rhUG; and
f. recovering the purified rhUG.
56. The method of claim 55, wherein the synthetic gene expressed in
the bacterial cells comprises Seq. ID Nos. 1-4.
57. The method of claim 55, wherein the filtering of step c
comprises the use of a tangential flow filtration (TFF) system.
58. The method of claim 55, wherein the exchange column of step d
is a Macro Q anion exchange column.
59. The method of claim 55, wherein the host-derived proteins of
step e are separated with a Chelating Sepharose Fast Flow (CSFF)
resin column.
60. The method of claim 55, wherein the rhUG recovered in step f is
substantially free of aggregates.
61. A method of producing a pharmaceutical grade rhUG drug
substance comprising the steps of: a. providing a bacterial
expression system capable of expressing rhUG; b. inoculating a
fermenter with an inoculum comprising the bacterial expression
system to form a fermentation culture; c. adding an induction agent
to the fermentation culture to induce the expression of rhUG by the
bacterial expression system; d. harvesting the rhUG expressed in
step c; and e. purifying the rhUG harvested in step d, wherein the
purifying step comprises the use of at least one filtration step
and at least one exhange column.
62. An assay method for determining the potency of recombinant
human uteroglobin in a sample which comprises: (a) contacting a
sample containing recombinant human uterogloblin with phospholipase
A.sub.2, (b) introducing a labeled substrate to said sample, (c)
separating product from sample, and (d) determining level of
cleavage.
63. The method of claim 62, wherein the assay is used in
conjunction with a standard .sup.14C-labeled assay.
64. The method of claim 62, wherein the radiolabeled substrate is
1-stearoyl-2-[.sup.14C]arachidonyl phosphotidyl choline.
65. The method of claim 62, wherein the recombinant human
uteroglobin phospholipase A.sub.2 is added to a final concentration
of 2 nM to 200 nM.
66. The method of claim 62, wherein the sample of step (a) is
preincubated for 15 minutes to 30 minutes at 30.degree. C. to
40.degree. C.
67. The method of claim 62, wherein the reaction in step (b) is
stopped by addition of an organic phase stopping solution.
68. The method of claim 62, wherein the sample in step (c) is
separated by vortexing and centrifugation.
69. The method of claim 62, wherein the product of step (c) is
separated from the sample by liquid-liquid separation.
70. The method of claim 62, wherein the level of cleavage in step
(d) is determined by scintillation counting.
71. A method for measuring in vitro the anti-inflammatory effect
arising from inhibition or blocking of secretory phopsholipase
A.sub.2 enzymes by recombinant human uteroglobin, comprising: (a)
contacting a sample containing recombinant human uterogloblin with
phospholipase A.sub.2, (b) introducing labeled substrate to said
sample, (c) separating product from sample, and (d) determining
level of cleavage by scintillation counting.
72. An assay method for assaying for the inhibition of secretory
phopsholipase A.sub.2 activity by recombinant human uteroglobin,
comprising: (a) contacting a sample containing recombinant human
uterogloblin with phospholipase A.sub.2, (b) introducing labeled
substrate to said sample, (c) separating product from sample, and
(d) determining level of cleavage by scintillation counting.
73. An assay method for determining the potency of recombinant
human uteroglobin in a sample which comprises: (a) contacting a
sample containing recombinant human uterogloblin with phospholipase
A.sub.2, (b) introducing flourescently labeled substrate to said
sample, (c) separating non-cleaved substrate from sample, and (d)
determining amount of cleaved substrate by flourescence.
74. The method of claim 73, wherein the sample of recombinant human
uteroglobin in step (a) has a final concentration of 34 nM to 34
.mu.M.
75. The method of claim 73, wherein the sample of step (a) is
preincubated for about 15 to 30 minutes at about 30 to 40.degree.
C.
76. The method of claim 73, wherein the flourescently-labeled
substrate is
2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indac-
ene-3propionyl)amino)undecyl)-sn-glycero-3-phosphotidylcholine.
77. The method of claim 73, wherein the reaction in step (b) is
stopped by addition of an organic phase stopping solution.
78. The method of claim 73, wherein in step (c) 1 .mu.L to 100
.mu.L of the stopped assay is loaded directly onto a silica normal
phase HPLC column.
79. A method for measuring in vitro the binding of recombinant
human uteroglobin to fibronectin, comprising: (a) contacting a
recombinant fragment of human fibronectin with a recombinant human
CC10-HRP conjugate, (b) visualizing the assay to determine binding
of recombinant human uteroglobin to the fibronectin fragment.
80. A method for determining the purity of recombinant human
uteroglobin which comprises, (a) taking samples of intermediates at
each step within the process of claim 35 and (b) analyzing the
process intermediates.
81. The method of claim 80, wherein process intermediates are
analyzed by SDS-PAGE.
82. The method of claim 80, wherein process intermediates are
analyzed by rhUG ELISA.
83. The method of claim 80, wherein process intermediates are
analyzed by LAL.
84. The method of claim 80, wherein process intermediates are
analyzed for protein content.
85. A pharmaceutical composition comprising the purified
recombinant human uteroglobin of claim 35.
86. A pharmaceutical composition comprising a purified recombinant
human uteroglobin and a pharmaceutically acceptable carrier or
diluent.
87. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin contains less than 5% aggregates of
recombinant human uteroglobin.
88. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin has a purity of greater than 95%.
89. The pharmaceutical composition of claim 86 wherein endotoxin
levels within said recombinant human uteroglobin comprises less
than 5 EU/mg rhUG.
90. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is in a sodium chloride solution.
91. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 4 months.
92. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 6 months.
93. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 9 months.
94. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 12 months.
95. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 15 months.
96. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 18 months.
97. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable is solution at 25.degree.
C. and 60% Room Humidity for at least 1 month.
98. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable is solution at 25.degree.
C. and 60% Room Humidity for at least 2 months.
99. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable is solution at 25.degree.
C. and 60% Room Humidity for at least 4 months.
100. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is stable is solution at 25.degree.
C. and 60% Room Humidity for at least 7 months.
101. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is safe to administer to a
mammal.
102. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is safe to administer to a human.
103. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is safe to administer via an
intratracheal tube.
104. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is safe to administer to a premature
infant.
105. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is safe to administer to a patient
receiving artificial surfactant.
106. The pharmaceutical composition of claim 86 wherein said
recombinant human uteroglobin is safe to administer to a patient in
respiratory distress.
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/898,616, filed Jul. 2, 2001, which is a continuation-in-part of
U.S. Ser. No. 08/864,357, filed May 28, 1997.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the production of
recombinant human uteroglobin (rhUG) which has use as a therapeutic
in the treatment of inflammation and fibrotic diseases, has
immunomodulatory effects, and regulates smooth muscle contraction.
More particularly, the invention provides processes, including
broadly the steps of bacterial expression and protein purification,
for the scaled-up production of rhUG according to current Good
Manufacturing Practices (cGMP). The invention further provides
analytical assays for evaluating the relative strength of in vivo
biological activity of rhUG produced via the scaled-up cGMP
processes.
BACKGROUND OF THE INVENTION
[0003] Therapeutic Uses of Recombinant Uteroglobin
[0004] The search for improved therapeutic agents for the treatment
of inflammatory, as well as fibrotic diseases, has received much
attention in recent years. Neonatal Respiratory Distress Syndrome
(RDS), a lung surfactant deficiency disease, is a condition of
particular interest in that it is one of the major causes of
mortality in premature neonates. While introduction of surfactant
therapy dramatically improves survival of RDS patients, the
development of chronic inflammatory and fibrotic disease in a
significant percentage of this patient population is a major
problem. Likewise, glomerular nephropathy and renal fibrosis leads
to end stage renal failure when patients' kidneys become blocked
and no longer filter the blood. Many forms of glomerular
nephropathy and renal fibrosis is characterized by fibronectin
deposits. In both diseases, fibronectin and collagen deposition and
fibrosis render the organ nonfunctional, and eventually, unable to
support life. Thus, these patients require chronic hemodialysis or
kidney transplantation.
[0005] Recombinant human UG is a protein with beneficial
anti-inflammatory, anti-fibrotic, anti-tumor, respiratory and
immunomodulatory properties that is under development as a
therapeutic agent in several clinical indications. Recombinant
human UG is useful for the treatment of conditions characterized by
a deficiency of UG. It is especially adapted for the treatment of
pulmonary inflammatory conditions, for example neonatal respiratory
distress syndrome (RDS) and bronchopulmonary dysplasia (BPD); for
the treatment of conditions characterized by an elevation in local
serum PLA.sub.2 activity, such as adult RDS (ARDS), septic shock,
pancreatitis, collagen vascular diseases, rheumatoid arthritis,
acute renal failure, and autoimmune uveitis; for the treatment of
conditions characterized by local elevations in PLA.sub.2 activity,
such as neonatal RDS/BPD, ARDS, rheumatoid arthritis, asthma,
peritonitis, glomerulopathies, including hereditary Fn-deposit
glomerulonephritis, and autoimmune uveitis; for the treatment of
fibrotic conditions where deposition of fibronectin is a causative
factor e.g., idiopathic pulmonary fibrosis, bleomycin lung, and
cystic fibrosis and glomerular nephropathy, particularly familial
glomeruleropathy, characterized by Fn deposits in the kidneys,
which ultimately lead to renal failure, can also be treated with
exogenous UG; and to methods for treating or preventing an
inflammatory or fibrotic condition characterized by a deficiency of
endogenous functional UG, by administering a compensating amount of
rhUG.
[0006] RhUG is useful for inhibiting cellular adhesion to
fibronectin, inhibits inflammatory cell and fibroblast migration on
already deposited fibronectin, and inhibits the interaction between
a cell and an extracellular matrix protein and/or membrane bound
protein. RhUG is also especially useful for improving and/or
normalizing lung function, pulmonary compliance, blood oxygenation,
and/or blood pH. RhUG is particularly useful in the regulation of
smooth muscle concentration in various organ systems including the
respiratory system, the digestive system, the circulatory system,
the reproductive system, and the urinary system. RhUG may also be
used as well to regulate or reduce vascular permeability, to
inhibit the migration of vascular endothelial cells and
angiogenesis, and to prevent angiogenesis. Intratracheal rhUG may
be used as a stem cell factor to increase lymphocyte production
and/or decrease polymorphonuclear leukocyte proliferation in the
long term. RhUG increases the concentration of circulating
lymphocytes and/or cytotoxic T cells while decreasing the
concentration of circulating polymorphonuclear leukocyte
proliferation, which is especially useful for patients suffering
from an autoimmune disease or allergy. Intravenous rhUG may be used
as well to suppress ATP metabolism in circulating lymphocytes and
to increase ATP metabolism in activated neutrophil, monocytes,
macrophages, and NK cells in the short term.
[0007] Prior Art Methods for the Production of Recombinant Human
Uteroglobin
[0008] There are several published methods for expressing rhUG and
for purifying either native or recombinant uteroglobin, or urine
protein-1, in microgram to milligram quantities for research
purposes (Mantile, 1993; Miele, 1992; Singh, 1987; Jackson, 1989;
Anderson, 1994; Umland, 1994; Aoki, 1996). These methods are quite
varied but none are well suited to large-scale production of a
protein and none address the regulatory issues required of a
process for production of a pharmaceutical. Furthermore, the
biological activities of these various preparations are not
necessarily equivalent. For example, Nieto (1997) reported that
native rabbit uteroglobin loses some of its progesterone activity
upon lyophilization, while Miele and Mantile use repeated size
exclusion chromatography steps and multiple lyophilizations as
concentration steps during their purification process. However, end
users would greatly prefer a ready-to-use product over a
lyophilized product since the percentage of aggregates of rhUG
increases with both lyophilization and repeated freeze thaw cycles.
High levels of aggregation can adversely affect the biological
activity, change the immunogenicity, or alter the potency of the
final drug product. Under FDA guidelines undesirable aggregates
constitute an impurity, entire lots of drug product may be rejected
on the basis of high levels of aggregate within the drug
product.
[0009] Problems in Development of Recombinant Therapeutics
[0010] The production of the recombinant protein-based drug
substances involves the development of several processes that
adhere to the guidelines set forth by the United States Food and
Drug Administration (FDA) referred to as current Good Manufacturing
Practices (cGMP). A process that adheres to the FDA's cGMP
guidelines is compliant with cGMP. In order to sell a
pharmaceutical composition or drug product in the U.S. and
elsewhere, it is necessary to produce the drug product using a cGMP
process.
[0011] The clinical development of a recombinant protein as a drug
substance, as well as the sales and use of protein drugs, require a
well-characterized and reproducible production process for the drug
substance as well as a detailed characterization of that drug
substance.
[0012] Recombinant proteins represent a particular challenge since
their activity is dependent not only upon amino acid composition
but also upon the conformation of the protein. The conformation of
a protein is the overall three-dimensional structure of the protein
which may be characterized on four levels. The first level is its
primary structure or amino acid sequence. The second level is the
protein's secondary structure and is the pattern of organization
associated with short stretches of about 6-30 amino acids in the
protein which form local stable structural regions such as alpha
helices, beta sheets, and omega loops. The third level, tertiary
structure consists of the groupings of secondary structures into
units or domains within a single contiguous stretch of amino acids,
representing a protein or peptide monomer. The four helical bundle
or fibronectin Type III repeat are examples of tertiary structures.
The fourth level, quaternary structure is present when two or more
individual peptide or protein monomers combine, either covalently
or non-covalently, to form a single functional unit.
[0013] Recombinant proteins present a further challenge since
activity is also dependent upon surface characteristics in which
charge and hydrophobic character, in addition to shape, contribute
significantly to the ability of a recombinant protein to interact
specifically with other biological and chemical substances in a
physiological environment. Isoforms of a protein consist of small
variations in conformation, surface charge and/or hydrophobic
character. These variations may result from changes in temperature,
or from interactions with chemicals, salts, or other biological
molecules (e.g., proteins, carbohydrates, lipids, nucleic acids,
etc.) in the surrounding environment, or from actual chemical
modifications to individual amino acids in the protein. Different
isoforms of a protein can be detected by high-resolution analytical
methods such as hydrophobic interaction HPLC, mass spectrometry,
capillary electrophoresis, peptide mapping, isoelectric focusing,
and two-dimensional electrophoresis.
[0014] The physical form and conformation of a recombinant protein
drug can be strongly influenced by the expression system in which
it is produced, as well as by the process through which it is
purified for use as a drug substance, for example. Likewise, the
resulting isoform or isoforms of the recombinant protein product
can also be strongly influenced by the expression system and
process through which it is produced. The biological activity of a
protein is highly dependent upon its conformation and isoform(s),
not just its chemical composition. For example, a protein may be
partially or completely denatured by exposure to high or low
temperatures and rendered biologically inactive, yet it still
retains the same sequence of amino acids. Therefore, the biological
activity of a recombinant protein is dependent not only on its
chemical composition, but also upon the process through which it is
expressed, purified, formulated and even packaged.
[0015] Drug substances or products often have extra components in
addition to the biologically active compound and its vehicle or
carrier. These components are derived from the raw materials from
which the drug was produced or from materials introduced as part of
the purification, formulation, or final packaging processes. The
drug substance is defined as the final form of purified bulk drug
while the drug product is the final packaged formulation of the
drug substance (e.g., the product used in the patient.) These extra
components of the drug substance or product are considered
impurities or contaminants and may have unintended or undesirable
biological activities of their own, either alone or in combination
with the drug itself. Contaminants may be defined as components
that are not derived from the drug itself while impurities may be
defined as components that contain some element of the drug itself
(e.g., fragments, variations, isoforms, enantiomers, aggregates,
etc.). Thus, the drug production process is important not only
because it determines the characteristics of the drug itself, but
also because it determines the level and nature of contaminants and
impurities in the drug substance and drug product. It is essential,
therefore, to carefully define the process in order to maintain
consistent and reproducible biological activity, in vivo, of a drug
substance, drug product, or pharmaceutical composition. Thus, the
process through which a recombinant protein drug is produced should
be sufficiently well-characterized so that it is capable of
complying with pharmaceutical production regulatory guidelines in
order to be commercially viable, since non-compliance results in a
product that cannot be sold or used in the U.S and elsewhere.
[0016] Moreover, the biopharmaceutical production process must be
sufficiently efficient and economical to be commercially viable.
Purification methods that are used in the laboratory to produce
small amounts of a protein for research purposes are not typically
suitable for biopharmaceutical production. For example, a small
scale method such as size exclusion chromatography often is not
practical for larger scale production because the chromatography
matrix would be crushed under its own weight in the size of column
required for purification of even a few grams of protein.
Furthermore, size exclusion chromatography always increases the
volume of the sample, resulting in less manageable high volume
purification intermediates that must be concentrated prior to the
next step in the process. Therefore, it is highly desirable to
avoid the use of size exclusion chromatography in a
biopharmaceutical production process. Another technique frequently
employed to preserve a protein pharmaceutical agent in a stable
form is lyophilization. This process involves the simultaneous
freeze-drying of a protein, converting it from a liquid form in
which it is typically susceptible to degradation, to a dry powder
form in which it can typically be stored for many months without
losing biological activity. However, repeated freeze/thaw cycles
increase the percentage of aggregates of rhUG, which may result in
a significant change in biological activity.
OBJECTS OF THE INVENTION
[0017] It is a primary object of the invention to provide a
bacterial expression system for the production of rhUG.
[0018] It is a further object of the invention to provide methods
for the production of rhUG and the purification of human
uteroglobin for substances suitable for use as a pharmaceutical
substance.
[0019] It is still a further object of the invention to provide
scaled-up production of rhUG conforming to cGMP standards.
[0020] A further and related object of the invention is to provide
methods to measure biological activities of human UG in vitro.
[0021] It is still a further object of the invention to provide
pharmaceutical preparations of human uteroglobin which are
commercially viable.
[0022] A further and related object of the invention is to provide
a method of producing rhUG research seed banks, master cell banks,
and production cell banks.
SUMMARY OF THE INVENTION
[0023] The invention provides a bacterial expression system for the
production of rhUG comprising a synthetic gene which codes for
human UG, wherein the synthetic gene comprises Seq. ID. Nos. 1-4.
The invention also provides a bacterial expression system for
production of rhUG comprising a human cDNA sequence which codes for
human UG wherein the gene further comprises Met-Ala-Ala at the
N-terminus of the sequence.
[0024] The invention further provides a method of producing a rhUG
research seed bank comprising: (a) inoculating onto a growth medium
at least one colony of a bacterial strain comprising a rhUG
expression system; (b) incubating the inoculated growth medium
until a stationary phase is reached; (c) adding glycerol to the
inoculated growth medium; (d) freezing the culture in aliquot
portions; and (e) storing the frozen aliquot portions at a
temperature below about -50 C.
[0025] The invention also provides a method of producing a rhUG
master cell bank comprising: (a) inoculating a suitable incubating
broth with an aliquot portion of a rhUG research seed bank; (b)
incubating the inoculated broth; (c) adding a cryopreservative to
the incubated broth to form a cryopreserved solution; (d)
transferring a portion of the cryopreserved solution to a cryovial;
and (e) storing the cryovial at a temperature below about -60
C.
[0026] The invention also provides a method of producing a rhUG
production cell bank comprising: (a) inoculating a suitable
incubating broth with an portion of a rhUG master cell bank; (b)
incubating the inoculated broth; (c) adding a cryopreservative to
the incubated broth to form a cryopreserved solution; (d)
transferring a portion of the cryopreserved solution to a cryovial;
and (e) storing the cryovial at a temperature below about -60
C.
[0027] The invention also provides a method of expressing rhUG
comprising the steps of: (a) providing a production seed cell bank
culture comprising an expression vector capable of expressing rhUG;
(b) inoculating a broth medium with the production seed cell bank
culture to form an inoculum; (c) incubating the inoculum formed in
step b; (d) inoculating a large scale fermenter with the inoculum
formed in step (c) to form a fermentation culture; (e) incubating
the fermentation culture within the large scale fermenter; (f)
adding an induction agent to the fermentation culture to induce the
expression of rhUG; and (g) harvesting the fermentation culture
after step (f).
[0028] The invention further provides a method of expressing rhUG
comprising the steps of: (a) inoculating a large scale fermenter
with an inoculum comprising an expression vector capable of
expressing rhUG to form a fermentation culture; (b) incubating the
fermentation culture within the large scale fermenter; (c) adding
an induction agent to the fermentation culture to induce the
expression of rhUG; and (d) harvesting the fermentation
culture.
[0029] The invention further provides a method of purifying rhUG
comprising the steps of: (a) providing a bacterial cell paste
comprising bacterial cells capable of overexpressing rhUG; (b)
lysing the bacterial cell paste to form a supernatant; (c)
filtering the supernatant formed in step b through a first nominal
molecular weight cut off (NMWCO) membrane to form a first permeate;
(d) concentrating the first permeate formed in step (c) by use of a
second NMWCO membrane to form a first concentrate; (e) loading the
concentrated permeate formed in step (d) onto an anion exchange
column to form a first eluate; (O) concentrating the first eluate
formed in step (e) by use of a third NMWCO membrane to form a
second concentrate; (g) loading the second permeate formed in step
(f) onto a Hydroxyapatite (HA) column to form a second eluate; (h)
separating host-derived proteins from the rhUG in the second eluate
formed in step (g) to provide purified rhUG; and (i) recovering the
purified rhUG formed in step (h).
[0030] The invention also provides a method of purifying rhUG
comprising the steps of: (a) providing bacterial cells capable of
overexpressing rhUG; (b) lysing the bacterial cells to form a
supernatant liquid; (c) filtering the liquid through a molecular
weight cut off (NMWCO) membrane; (d) loading the liquid onto an
exchange column; (e) separating host-derived proteins from the rhUG
to provide purified rhUG; and (f) recovering the purified rhUG.
[0031] The invention provides a method of producing a
pharmaceutical grade rhUG drug substance comprising the steps of:
(a) providing a bacterial expression system capable of expressing
rhUG; (b) inoculating a fermenter with an inoculum comprising the
bacterial expression system to form a fermentation culture; (c)
adding an induction agent to the fermentation culture to induce the
expression of rhUG by the bacterial expression system; (d)
harvesting the rhUG expressed in step (c); and (e) purifying the
rhUG harvested in step (d), wherein the purifying step comprises
the use of at least one filter and at least one exhange column.
[0032] The invention provides an assay method for determining the
potency of recombinant human uteroglobin in a sample which
comprises: (a) contacting a sample containing recombinant human
uterogloblin with phospholipase A.sub.2, (b) introducing a
radiolabeled substrate to said sample, (c) separating product from
sample, and (d) determining level of cleavage.
[0033] The invention further provides a method for measuring in
vitro the anti-inflammatory effect arising from inhibition or
blocking of secretory phopsholipase A.sub.2 enzymes by recombinant
human uteroglobin, comprising: (a) contacting a sample containing
recombinant human uterogloblin with phospholipase A.sub.2, (b)
introducing radiolabeled substrate to said sample, (c) separating
product from sample, and (d) determining level of cleavage by
scintillation counting.
[0034] The invention also provides an assay method for assaying for
the inhibition of secretory phopsholipase A.sub.2 activity by
recombinant human uteroglobin, comprising: (a) contacting a sample
containing recombinant human uterogloblin with phospholipase
A.sub.2, (b) introducing radiolabeled substrate to said sample, (c)
separating product from sample, and (d) determining level of
cleavage by scintillation counting.
[0035] The invention also provides an assay method for determining
the potency of recombinant human uteroglobin in a sample which
comprises: (a) contacting a sample containing recombinant human
uterogloblin with phospholipase A.sub.2, (b) introducing
flourescently labeled substrate to said sample, (c) separating
non-cleaved substrate from sample, and (d) determining amount of
cleaved substrate by flourescence.
[0036] The invention provides a method for measuring in vitro the
binding of recombinant human uteroglobin to fibronectin,
comprising: (a) contacting a recombinant fragment of human
fibronectin with a recombinant human CC10-HRP conjugate, (b)
visualizing the assay to determine binding of recombinant human
uteroglobin to the fibronectin fragment.
[0037] The invention also provides a pharmaceutical composition
comprising a purified recombinant human uteroglobin and a
pharmaceutically acceptable carrier or diluent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The invention will now be described in more detail, with
reference to the accompanying drawings, in which:
[0039] FIG. 1 shows construction of synthetic bacterial gene for
rhUG.
[0040] FIG. 2 shows expression of rhUG using a synthetic bacterial
gene: SDS-PAGE analysis.
[0041] 10-20% Tricine Gel: Lanes are from left to right: Lane 1:
Size Standard Lane 2: Uninduced bacterial lysate, Lane 3: Induced
bacterial lysate.
[0042] FIG. 3 shows the genetic map of plasmid pCG12.
[0043] FIG. 4 shows a flowchart of master and production seed cell
banking process.
[0044] FIG. 5 shows growth curves of bacterial cultures from which
master and production seed cell banks were derived.
[0045] Cell growth was followed by Optical Density at 600 nm for
both the Master (1) and Production (n) seeds.
[0046] FIG. 6 shows a flowchart of fermentation process for rhUG
expression.
[0047] FIG. 7 shows a growth curve of fermentation culture.
[0048] Culture growth was followed by the OD at 600 nm (O),
aeration (DO, .circle-solid.) was followed by a dissolved oxygen
probe, and agitation (.box-solid.) was followed as a function of
the rpm's.
[0049] FIG. 8 shows SDS-page analysis of rhUG expression during
fermentation.
[0050] 10-20% Tricine gel. Lanes are from left to right, Lane 1,
Rainbow Standard; the fermentation samples taken at the indicated
times, post-induction: lane 2, 3.8 hr; lane 4.0 hr; lane 4, 4.2 hr;
lane 5, 4.5 hr; lane 6, 5.0 hr; lane 7, 5.6 hr; and lane 8, 6
hr.
[0051] FIG. 9a and b show flow diagram of purification scheme, and
minor variations thereof.
[0052] FIG. 10 shows a detailed flow diagram of the initial TFF and
diafiltration.
[0053] FIG. 11a shows a detailed flow diagram of the Macro Q anion
exchange chromatography step.
[0054] FIG. 11b shows a representative chromatogram of the Macro Q
anion exchange chromatography step.
[0055] FIG. 12 shows a detailed flow diagram of the second
concentration/diafiltration step.
[0056] FIG. 13a shows a detailed flow diagram of the hydroxyapatite
chromatography step.
[0057] FIG. 13b shows a representative chromatogram of the
hydroxyapatite chromatography step.
[0058] FIG. 14a shows a detailed flow diagram of the copper
chelation chromatography step.
[0059] FIG. 14b shows a representative chromatogram of the copper
chelation chromatography step.
[0060] FIG. 15 shows a detailed flow diagram of Sartobind Q and
third concentration/diafiltration step.
[0061] FIG. 16 shows a detailed flow diagram of final diafiltration
and formulation.
[0062] FIG. 17 shows purification of rhUG by cation exchange
chromatography.
[0063] FIG. 18 shows purification of rhUG by HIC
chromatography.
[0064] FIG. 19 shows a standard curve for competitive ELISA for
UG.
[0065] FIG. 20 shows a chromatogram of SPLA2 assay.
[0066] FIG. 21 shows a standard curve for fibronectin binding
assay.
[0067] FIG. 22a show assessment of purification steps by
SDS-PAGE.
[0068] 10-20% Tricine gel, samples are, from left to right: lane 1,
Rainbow Standard; lane 2, Crude lysate; lane 3, 100 K Retentate;
lane 4, 5 K Ret; Lane 5, #1, Macro Q Passthrough; lane 6, Macro Q
Wash I#1; lane 7, Macro Q Wash I#2; lane 8, Macro Q Wash 1#3; and
lane 9, Macro Q eluate.
[0069] FIG. 22b: Assessment of Purification Steps by SDS-PAGE.
[0070] 10-20% Tricene gel, samples are, from left to right: lane 1,
Rainbow Standard; lane 2, Hydroxyapatite Passthrough; lane 3,
Hydroxyapatite Wash I; lane 4, Hydroxyapatite eluate; lane 5,
Copper CSFF Passthrough; lane 6, Sartobind Q Passthrough; lane 7,
Purified rhCC10 Bulk.
[0071] FIG. 23 shows SDS-PAGE analysis of purity of drug
substance.
[0072] 10-20% Tricine gel: samples are, from left to right Lane 1,
Rainbow Standard; lane 3, 5 .mu.g 0726; lane 5, 5 .mu.g reduced
0726; lane 7, 10 .mu.g 0726; lane 9, 10 .mu.g reduced 0726; lane
11, 55 .mu.g 0726. Lanes 2, 4, 6, 8, 10, and 12 were left
unfilled.
[0073] FIG. 24 shows Western Blot of drug substance using anti-UG
polyclonal antibody.
[0074] 10-20% Tricine gel transferred to Hybond-P PVDF transfer
membranes, samples are, from left to right: Lane 1, Rainbow
standard;
[0075] lane 2, rhCC10 (lot 0726); lane 3, rhCC10 (lot 0728) lane 4,
Rainbow Standard; lane 5, reduced rhCC10 (lot 0726); Lane 6,
reduced rhCC10 (lot 0728).
[0076] FIG. 25 shows Western Blot of drug substance using anti-E.
coli lysate polyclonal antibody.
[0077] 10-20% Tricine gel transferred to Hybond-P PVDF transfer
membranes, Samples are, from left to right: Lane 1, Standard, E.
coli cell lysate from BL21 (DE3); lane 2, Rainbow Standard; lane 3,
rhCC10 (lot 0726); Lane 4, rhCC10 (lot 0728). The arrow indicates
the location of the E. coli impurity in lanes 3 and 4.
[0078] FIG. 26 shows isoelectric focusing PAGE gel of rhUG drug
substance.
[0079] Samples are, from left to right: lane 1, standards; lane 2,
55 .mu.g of rhCC10 lot 0726; lane 3, 55 .mu.g of rhCC10 lot
rhCC10/.
[0080] FIG. 27 shows a flow diagram of rhUG fill process.
[0081] FIG. 28 shows SDS-PAGE analysis of purity of drug
product.
[0082] 10-20% Tricine gel, samples are, from left to right: lane 1,
Rainbow Standard; lane 3, 5 FIG. 0728; lane 5, 5 .mu.g reduced
0728; lane 7, 10 .mu.g 0728; lane 9, 10 .mu.g reduced 0728; lane
11, 10 .mu.g reduced research control, rhCC10/7. Lanes 2, 4, 6, 8,
10, and 12 were left unfilled.
[0083] FIG. 29 shows Western Blot of drug product using anti-UG
polyclonal antibody.
[0084] 10-20% Tricine gel transferred to Hybond-P PVDF transfer
membranes, samples are, from left to right: lane 1, Rainbow
standard;
[0085] lane 2, rhCC10 (lot 0726); lane 3, rhCC10 (lot 0728); lane
4, Rainbow Standard; lane 5, rhCC10 (lot 0726); lane 6, reduced
rhCC10 (lot 0728).
[0086] FIG. 30 shows Western Blot of drug product using anti-E.
coli lysate polyclonal antibody.
[0087] 10-20% Tricine gel transferred to Hybond-P PVDF transfer
membranes, samples are, from left to right: lane 1, Standard, E.
coli cell lysate from BL21 (DE3); lane 2, Rainbow Standard; lane 3,
rhCC10 (lot 0726); lane 4, rhCC10 (lot 0728). The arrow indicates
the position of the band at 40 kD in lanes 3 and 4.
[0088] FIG. 31 shows isoelectric focusing PAGE gel of rhUG drug
product.
[0089] Samples are, from left to right, lane 1, standards; lane 2,
55 .mu.g of rhCC10 lot 0728; lane 3, 55 .mu.g of rhCC10 lot
rhCC10/7.
[0090] FIG. 32 shows the nucleotide sequence for cCG12 (SEQ. ID NO.
9)
[0091] FIG. 33 shows the amino acid sequence for rhUG (SEQ. ID NO.
10)
DETAILED DESCRIPTION OF THE INVENTION
[0092] The disclosures in copending application Ser. Nos.
08/864,357; 09/087,210; 09/120,264; 09/549,926; 09/861,688;
PCT/US98/11026 (WO 98/53846); PCT/US99/16312 (WO ______);
PCT/US00/09979 (WO ______); and PCT/US01/12126 (WO ______) are
hereby incorporated by reference.
[0093] Definitions
[0094] "Pure rhUG" as used herein means 1) that no other proteins
are detectable in the rhUG preparation by SDS-PAGE, Western blot or
immunoprecipitation with anti-E. coli antibodies, or by analytical
HPLC; 2) that no bacterial endotoxin is detectable by LAL test; 3)
that no bacterial nucleic acid is detectable by Southern blot (DNA
hybridization).
[0095] "Purified rhUG" as used herein means rhUG which has met all
specifications relating to purity as defined herein.
[0096] "Pharmaceutical Grade rhUG" as defined herein means rhUG
which as met all purity, physical and biological activity
specifications as defined herein and described in application Ser.
Nos. 08/864,357; 09/087,210; 09/120,264; 09/549,926; 09/861,688;
PCT/US98/11026; PCT/US99/16312; PCT/US00/09979; and
PCT/US01/12126.
[0097] "Isoforms" as used herein refers to alternative forms of a
protein that can be distinguished by physical or chemical means and
may possess different biological activities, including different
conformations, small variations in chemical composition of amino
acids resulting from post-translational modifications, or
variations in purification and processing.
[0098] "Conformation" as used herein refers to the
three-dimensional structure of a protein, including the way it is
folded, surface charge and hydrophobicity distribution. Any given
protein may have several conformations that can affect its
interactions with the surrounding environment, as well as other
proteins, chemicals and cells.
[0099] "Aggregates" as used herein refers to complexes made up of
multiple individual units of a single protein.
[0100] "Impurity" as used herein refers to compounds routinely
present in the final product other than the drug or biologic of
interest and required excipients; impurities can be either product
or process related.
[0101] "Contaminant" as used herein refers to compounds or
materials present in the final product that are not routinely
present.
[0102] "Specifications" as used herein refers to a set of criteria
that define a pharmaceutical grade protein, drug substance and drug
product with respect to physical and chemical parameters, as well
as biological activity and antigenic identity.
[0103] "Potency Assay" as used herein refers to a specific test
that is used to measure the biological activity of drug substance
or drug product and is used to gauge the strength of the biological
activities of the drug in vivo, for purposes of relating biological
activity to a physical parameter such as protein concentration and
comparing different preparations of a given drug to each other.
[0104] "Immunoassay" as used herein refers to a test that is used
to identify an antigen, in this case uteroglobin, through the use
of one or more antibodies, including ELISAs (enzyme-linked
immunosorbent assay).
[0105] "Formulation" as used herein refers to a specific
pharmaceutical composition containing the biologically active drug
substance plus specific excipients.
[0106] "Process Intermediate" as used herein refers to a sample
comprising or derived from the product of each step in a
process.
[0107] "Bulk Drug"--(See "Drug Substance")
[0108] "Drug Substance" as used herein refers to the purified drug,
i.e. rhUG, prior to final formulation and fill into the final drug
containers.
[0109] "Drug Product" as used herein refers to the drug in its
final form for use in the patient population.
[0110] "RhUG Drug Substance" as used herein means pharmaceutical
grade preparation of rhUG meeting specifications set forth herein
and mediates the activity described herein and in application Ser.
Nos. 08/864,357; 09/087,210; 09/120,264; 09/549,926; 09/861,688;
PCT/US98/11026; PCT/US99/16312; PCT/US00/09979; and
PCT/US01/12126.
[0111] "RhUG Drug Product" as used herein means pharmaceutical
grade preparation of rhUG meeting specifications set forth herein
and mediates the activity described herein and in application Ser.
Nos. 08/864,357; 09/087,210; 09/120,264; 09/549,926; 09/861,688;
PCT/US98/11026; PCT/US99/16312; PCT/US00/09979; and
PCT/US01/12126.
[0112] "Standard Operating Procedure" as used herein means a
defined procedure for the execution of a particular task under cGMP
guidelines.
[0113] "Research Seed Bank" as used herein means a seed bank made
under GLP conditions.
[0114] "Master Seed Bank" as used herein means a seed bank made
under cGMP conditions, the purpose of the seed bank is to act as a
source for the production of the Production Seed bank and for long
term storage.
[0115] "Production Seed bank" as used herein means a seed bank made
under cGMP conditions to be used to initiate the cGMP
fermentation.
[0116] "cGMP" as used herein means current Good Manufacturing
Practices.
[0117] "cGMP Production Process" as used herein means a production
process that occurs under cGMP as defined by the FDA.
[0118] "Stable Drug Substance/Product" as used herein means a drug
substance/product that meets a series of pre-defined criteria
indicative of biological and physical stability.
[0119] "Biologically Active rhUG" as used herein means UG which can
both inhibit the activity of PLA.sub.2 and bind to recombinant
human fibronectin fragments, and activities described in
application Ser. Nos. 08/864,357; 09/087,210; 09/120,264;
09/549,926; 09/861,688; PCT/US98/11026; PCT/US99/16312;
PCT/US00/09979; and PCT/US01/12126.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0120] The invention includes bacterial expression systems for the
production of rhUG. In one embodiment the bacterial expression
system comprises a synthetic gene which codes for human UG. In
another embodiment the synthetic gene comprises Seq. ID. Nos. 1-4.
The invention also provides a bacterial expression system for
production of rhUG comprising a human cDNA sequence which codes for
human UG wherein the gene further comprises Met-Ala-Ala at the
N-terminus of the sequence. In a further embodiment the synthetic
gene further comprises Met-Ala-Ala at the N terminus of the
synthetic gene. In still another embodiment the expression system
further comprises an approximately 2.8 kb par sequence.
[0121] The invention also includes methods of producing a rhUG
research seed bank. In one embodiment the method of producing a
rhUG research seed bank comprises the steps of: a) inoculating onto
a growth medium at least one colony of a bacterial strain
comprising a rhUG expression system; b) incubating the inoculated
growth medium until a stationary phase is reached; c) adding a
cryopreservative, e.g. glycerol, to the inoculated growth medium;
d) freezing the culture in aliquot portions; and e) storing the
frozen aliquot portions at a temperature below about -50 C,
preferably from -50.degree. C. to -100.degree. C. In another
embodiment the method of producing a rhUG research seed bank
comprises incubating the inoculated growth medium, monitoring the
growth by optical density (OD) from 550 nm to 660 run, preferably
at 600 nm, until an optical density of about 0.8 Absorbance Units
(AU) to 1.5 AU is reached.
[0122] The invention also includes methods of producing a rhUG
master cell bank comprising the steps of: a) inoculating a suitable
incubating broth with an aliquot portion of a rhUG research seed
bank to form a bacterial culture; b) incubating the bacterial
culture; c) adding a cryopreservative to the bacterial culture to
form a cryopreserved solution; d) transferring a portion of the
cryopreserved solution to a cryovial; and e) storing the cryovial
at a temperature below about -60 C, preferably from -50.degree. to
-100.degree. C. In one embodiment the method of producing a rhUG
master cell bank comprises incubating the bacterial culture,
monitoring the growth by optical density (OD) from 550 nm to 660
nm, preferably at 600 nm, until an optical density of about 0.8 AU
to 1.5 AU is reached.
[0123] Methods for producing a rhUG production cell bank from a
portion of the master cell bank are also disclosed.
[0124] The present invention includes methods for expressing rhUG.
In one embodiment the method for expressing rhUG comprises the
steps of: a) providing a production seed cell bank culture
comprising an expression vector capable of expressing rhUG; b)
inoculating a broth medium with the production seed cell bank
culture to form an inoculum; c) incubating the inoculum formed in
step b; d) inoculating a large scale fermenter with the inoculum
formed in step (c) to form a fermentation culture; e) incubating
the fermentation culture formed in step (d) within the large scale
fermenter; f) adding an induction agent to the fermentation culture
formed in step (e) to induce the expression of rhUG; and harvesting
the fermentation culture.
[0125] In one embodiment the method for expressing rhUG uses an
expression vector comprising Seq. ID Nos. 1-4. In another
embodiment the inoculum is incubated for a period between about 12
hours and about 24 hours at a temperature between about 28.degree.
C. and about 36.degree. C. In yet another embodiment the incubation
of step (e) is continued until a minimum OD, in the range of 550 nm
to 660 nm, preferably at 600 nm, of two Absorbance Units is
reached.
[0126] The induction agent may be
isopropyl-beta-D-thiogalactopyranoside (IPTG). In still another
embodiment about 1 to 4 hours elapses between the induction step
and the harvesting step. In yet another embodiment harvesting the
fermentation culture utilizes centrifugation.
[0127] The present invention provides further methods of expressing
rhUG comprising the steps of: a) inoculating a large scale
fermenter with an inoculum comprising an expression vector capable
of expressing rhUG to form a fermentation culture; b) incubating
the fermentation culture within the large scale fermenter c) adding
an induction agent to the fermentation culture to induce the
expression of rhUG; and d) harvesting the fermentation culture.
[0128] In one embodiment for expressing rhUG the expression vector
comprises Seq. ID Nos. 1-4. The invention provides in another
embodiment the large scale fermenter has at least a 300 liter
capacity. In yet another embodiment the incubation of step b is
continued until a minimum optical density from 550 nm to 660 nm,
preferably 600 nm, of about 2.0 AU is achieved. In still another
embodiment the induction agent comprises
isopropyl-beta-D-thiogalactopyranoside (IPTG). In a further
embodiment about 1 to about 4 hours elapses between step c and step
d. In a further embodiment harvesting the fermentation culture
comprises centrifugation.
[0129] The invention further includes methods of purifying rhUG. In
one embodiment the method of purifying rhUG comprising the steps
of: a) providing a bacterial cell paste comprising bacterial cells
capable of overexpressing rhUG; b) lysing the bacterial cell paste
to form a supernatant; c) filtering the supernatant through a first
nominal molecular weight cut off (NMWCO) membrane to form a first
permeate; d), concentrating the first permeate by the use of a
second NMWCO membrane to form a first concentrate; e) loading the
concentrated permeate formed in step (d) onto an anion exchange
column to form a first eluate; f) concentrating the first eluate
formed in step (e) by the use of a third NMWCO membrane to form a
second concentrate; (g) loading the second concentrate onto a
Hydroxyapatite (HA) column to form a second eluate; h) separating
host-derived proteins in the second eluate, from the rhUG to
provide purified rhUG; and i) recovering the purified rhUG.
[0130] In one embodiment the method of purifying rhUG, utilizes
bacterial cells which comprise Seq. ID Nos. 1-4. In another
embodiment lysing the bacterial cell paste is achieved through
shearing. In still another embodiment cell debris is removed by
centrifugation between steps (b) and (c). In yet another embodiment
the membrane of step (b) is about a 30K to 100K NMWCO membrane.
[0131] In another embodiment the filtering of step (c) comprises
the use of a tangential flow filtration (TFF) system. In another
embodiment the membrane of step d is about a 5 k cutoff membrane.
In still another embodiment the anion exchange column is a Macro Q
anion exchange column. In yet another embodiment the host-derived
proteins are separated with a Chelating Sepharose Fast Flow (CSFF)
resin column. In one embodiment the CSFF resin column comprises
copper. In yet another embodiment the host-derived proteins are
separated from the rhUG by filtering the rhUG through a 30 K NMWCO
membrane.
[0132] In another embodiment a positively charged membrane is
placed downstream of the CSFF column forming a pass through
substantially free of host derived proteins. In one embodiment this
positively charged membrane is a Sartobind Q TFF membrane. In still
another embodiment the pass through is diafiltered through about a
5K NMWCO membrane. In another embodiment the rhUG recovered in step
i is substantially free of aggregates.
[0133] The present invention provides further methods of purifying
rhUG. One of these further methods comprises the steps of: a)
providing bacterial cells capable of overexpressing rhUG; b) lysing
the bacterial cells to form a supernatant liquid; c) filtering the
liquid through a molecular weight cut off (NMWCO) membrane; d)
loading the liquid onto an exchange column; e) separating
host-derived proteins from the rhUG to provide purified rhUG; and
f) recovering the purified rhUG.
[0134] In another embodiment the filtering of step c comprises the
use of a tangential flow filtration (TFF) system. In yet another
embodiment the anion exchange column is a Macro Q anion exchange
column. In still another embodiment the host-derived proteins are
separated with a Chelating Sepharose Fast Flow (CSFF) resin column.
In another embodiment the recovered rhUG is substantially free of
aggregates.
[0135] The present invention also provides methods of producing a
pharmaceutical grade rhUG drug substance comprising the steps of:
a) providing a bacterial expression system capable of expressing
rhUG; b) inoculating a fermenter with an inoculum comprising the
bacterial expression system to form a fermentation culture; c)
adding an induction agent to the fermentation culture to induce the
expression of rhUG by the bacterial expression system; d)
harvesting the rhUG expressed in step c; and e) purifying the rhUG
harvested in step d, wherein the purifying step comprises the use
of at least one filter and at least one exhange column.
[0136] The invention also includes an assay method for determining
the potency of recombinant human uteroglobin in a sample which
comprises: (a) contacting a sample containing recombinant human
uterogloblin with phospholipase A.sub.2, (b) introducing a labeled
substrate to said sample, and (c) separating product from sample,
and (d) determining level of cleavage. In one embodiment, the assay
is used in conjunction with a standard .sup.14C-labeled assay. In
another embodiment of the invention, the labeled substrate is
1-stearoyl-2-[1-.sup.14C]arachidonyl phosphatidyl choline. In a
further embodiment, the recombinant human phospholipase A.sub.2 is
added to a final concentration of from 2 nM to 200 nM in step (a).
In another embodiment of the invention, the sample of step (a) is
preincubated for from 15 minutes to 30 minutes at from 30.degree.
C. to 40.degree. C. In yet another embodiment of the invention, the
labeled substrate added in step (b) is added to a final
concentration of from 0.5 .mu.g/ml to 50 .mu.g/ml.
[0137] In an embodiment of the invention, the reaction in step (b)
is stopped after from 5 minutes to 30 minutes by addition of an
organic phase stopping solution. One example of an organic phase
stopping solution is a 7.7 dilution with Doles reagent and purified
water (84:16). In an embodiment of the invention, the sample in
step (c) is separated by vortexing and centrifugation, and the
product of step (c) is arachidonic acid, which is separated from
the sample by liquid-liquid separation in step (c).
[0138] In an embodiment of the invention, the sample is separated
and the top layer removed for scintillation counting to determine
the level of cleavage in step (d). Separation may be accomplished
by vortex and centrifugation.
[0139] The present invention also provides a method for measuring
in vitro the anti-inflammatory effect arising from inhibition or
blocking of secretory phopsholipase A.sub.2 enzymes by recombinant
human uteroglobin, comprising: (a) contacting a sample containing
recombinant human uterogloblin with phospholipase A.sub.2, (b)
introducing labeled substrate to said sample, (c) separating
product from sample, and (d) determining level of cleavage by
scintillation counting.
[0140] The present invention further provides an assay method for
assaying for the inhibition of secretory phopsholipase A.sub.2
activity by recombinant human uteroglobin, comprising: (a)
contacting a sample containing recombinant human uterogloblin with
phospholipase A.sub.2, (b) introducing labeled substrate to said
sample, (c) separating product from sample, and (d) determining
level of cleavage by scintillation counting.
[0141] The present invention provides an assay method for
determining the potency of recombinant human uteroglobin in a
sample which comprises: (a) contacting a sample containing
recombinant human uterogloblin with phospholipase A.sub.2, (b)
introducing flourescently labeled substrate to said sample, (c)
separating non-cleaved substrate from sample, and (d) determining
amount of cleaved substrate by flourescence.
[0142] In an embodiment of the invention, the sample of recombinant
human uteroglobin in step (a) has a final concentration of 34 nM to
34 .mu.m. In another embodiment of the invention, the sample of
step (a) is preincubated for 15-30 minutes at 30-40.degree. C.
[0143] In a further embodiment of the invention, the
flourescently-labeled substrate is
2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a--
diaza-s-indacene-3propionyl)amino)undecyl)-sn-glycero-3-phosphotidylcholin-
e. In yet another embodiment of the invention, the substrate added
in step (b) is added to a final concentration of 0.5-50
.mu.g/ml.
[0144] In an embodiment of the invention, the reaction in step (b)
is stopped after 5-30 minutes by addition of a one to five dilution
of an organic phase stopping solution. In another embodiment of the
invention the organic phase stopping solution is
2-Propanol:n-hexane (8:3). In another embodiment of the invention,
1 .mu.L to 100 .mu.L of the stopped assay is loaded directly onto a
silica normal phase HPLC column in step (c). In a further
embodiment of the invention, the flourescence of step (d) has
excitation at 460 nm to 505 nm and emmision at 505 nm to 550
nm.
[0145] The present invention provides a method for measuring in
vitro the binding of recombinant human uteroglobin to fibronectin,
comprising: (a) contacting a recombinant fragment of human
fibronectin with a recombinant human CC10-HRP conjugate, and (b)
visualizing the assay to determine binding of recombinant human
uteroglobin to the fibronectin fragment.
[0146] The present invention also provides a method for determining
the purity of recombinant human uteroglobin which comprises, (a)
taking samples of intermediates at each step within the process of
claim, and (b) analyzing the process intermediates.
[0147] In an embodiment of the invention, process intermediates are
analyzed by SDS-PAGE in step (b). In another embodiment of the
invention, process intermediates are analyzed by rhUG ELISA in step
(b). In a further embodiment of the invention, process
intermediates are analyzed by LAL in step (b). In yet another
embodiment of the invention, intermediates are analyzed for protein
content in step (b).
[0148] The present invention provides a pharmaceutical composition
comprising the purified recombinant human uteroglobin of the
present invention. The present invention also provides a
pharmaceutical composition comprising a purified recombinant human
uteroglobin and a pharmaceutically acceptable carrier or
diluent.
[0149] In an embodiment of the invention, the recombinant human
uteroglobin contains less than 5% aggregates of recombinant human
uteroglobin. In another embodiment of the invention, the
recombinant human uteroglobin has a purity of greater than 95%. In
a further embodiment of the invention, the level of endotoxin in
the recombinant human uteroglobin comprises less than 5 EU/mg rhUG.
In yet another embodiment of the invention, the recombinant human
uteroglobin is in a sodium chloride solution.
[0150] In an embodiment of the invention, the recombinant human
uteroglobin is stable in solution at 4.degree. C. for at least 4
months. In another embodiment of the invention, the recombinant
human uteroglobin is stable in solution at 4.degree. C. for at
least 6 months. In a further embodiment of the invention, the
recombinant human uteroglobin is stable in solution at 4.degree. C.
for at least 9 months. In yet another embodiment of the invention,
the recombinant human uteroglobin is stable in solution at
4.degree. C. for at least 12 months. In yet another embodiment of
the invention, the recombinant human uteroglobin is stable in
solution at 4.degree. C. for at least 15 months. In yet another
embodiment of the invention, the recombinant human uteroglobin is
stable in solution at 4.degree. C. for at least 18 months. In an
embodiment of the invention, the recombinant human uteroglobin is
stable in solution at 25.degree. C. and 60% room humidity for at
least 1 months. In an embodiment of the invention, the recombinant
human uteroglobin is stable in solution at 25.degree. C. and 60%
room humidity for at least 2 months. In an embodiment of the
invention, the recombinant human uteroglobin is stable in solution
at 25.degree. C. and 60% room humidity for at least 4 months. In an
embodiment of the invention, the recombinant human uteroglobin is
stable in solution at 25.degree. C. and 60% room humidity for at
least 7 months.
[0151] In another embodiment of the invention, the recombinant
human uteroglobin is safe to administer to a mammal. In a further
embodiment of the invention, the recombinant human uteroglobin is
safe to administer to a human. In yet another embodiment of the
invention, the recombinant human uteroglobin is safe to administer
via an intratracheal tube. In an embodiment of the invention, the
recombinant human uteroglobin is safe to administer to a premature
infant. In another embodiment of the invention, the recombinant
human uteroglobin is safe to administer to a patient receiving
artificial surfactant. In a further embodiment of the invention,
the recombinant human uteroglobin is safe to administer to a
patient in respiratory distress.
[0152] Recombinant human UG was produced by the procedures
described below. The rhUG had levels of purity exceeding 97% such
that it may be used, inter alia, according to the inventions
described in application Ser. Nos. 08/864,357; 09/087,210;
09/120,264; 09/549,926; 09/861,688; PCT/US98/11026; PCT/US99/16312;
PCT/US00/09979; and PCT/US01/12126 to modulate the immune response,
to inhibit inflammation and reduce or prevent fibrosis and
unregulated cell proliferation in vitro and in vivo. In certain
embodiments the level of purity was 99% or greater.
[0153] Preparation of the rhUG-Expressing Episome and the
rhUG-Producing Strain of Bacteria
[0154] Two types of novel bacterial expression systems were
developed for the production of recombinant human UG. One involved
the construction of a novel synthetic gene for human UG, using
codons optimized for bacterial protein synthesis. The other
provides for the use of a bacterial genetic element conferring
stable plasmid inheritance in the absence of antibiotic selection.
Both approaches yielded bacterial host-vector systems capable of
efficient UG overexpression.
[0155] Construction of a Synthetic Bacterial Gene for rhUG
[0156] A synthetic bacterial gene sequence for human UG was
designed to improve bacterial expression and was assembled from
synthetic oligonucleotides. Because mature native UG has a glutamic
acid residue at its N-terminus, an initiator methionine must be
added at the N-terminus, which allows initiation of peptide
synthesis (translation) from mRNA in bacteria. Codon usage was
optimized for expression in bacteria, by incorporating the most
frequently used codons in bacteria (Anderssen and Kurland, 1990)
into the protein coding sequence. Synthtetic genes for expression
of recombinant human UG can similarly be constructed by tailoring
codon usage for optimized expression in insect cells, plant cells,
yeast cells, and other non-primate mammalian species. The
optimization of codon usage results in a higher translation
efficiency and protein expression level. The use of codons
preferred in bacteria also may decrease the stress response to the
metabolic burden created by the consumption of rare charged tRNAs.
Without being bound by a particular theory, it is believed that
this stress response resulting from imbalances in bacterial charged
tRNAs may alter transcription patterns, increases cellular
proteolysis, and alters cellular metabolism. All of these factors
may contribute to problems with reproducibility in fermentation
cultures in which the protein product is expressed. Variations in
the production cultures may lead to larger problems in downstream
purification in which new bacterial proteins appear at different
steps in the process.
EXAMPLE I
Assembly of a Synthetic Bacterial Gene
[0157] The synthetic bacterial gene for rhUG was assembled from
oligonucleotides as shown in Table 1. (Synthetic oligonucleotides
were obtained from Bioserve Biotechnologies, Inc.) Oligonucleotides
1-4 (SEQ. ID NOS. 1-4), respectively, represent the coding strand
and 5-8 (SEQ. ID NOS. 5-8), respectively, represent the
complementary strand. Both sets are in order from 5' to 3',
respectively, and were assembled by annealing and ligation using
standard methods as shown in FIG. 1. Ligation mixtures were
transformed into appropriate strains as shown in Table 2 below and
plasmid vector-bearing colonies were selected with appropriate
antibiotic. Transformants were initially screened with a quick PCR
assay done directly on the bacterial colonies to determine insert
size. Colonies bearing the .about.234 bp rhUG insert were then
subcultured for further screening. The secondary screen was done on
10 ml bacterial cultures from PCR-positive clones, where expression
of an inducible protein band of approximately 10 kDa is sought (See
FIG. 2) Samples of whole cells, induced appropriately for
expression of rhUG, were directly lysed in 2.times.SDS-PAGE gel
loading buffer. The samples were run on 16% Tris-glycine SDS-PAGE
gels in a minigel apparatus (Novex). The screening procedures were
as described in detail in Pilon (1997), incorporated herein by
reference. Plasmid DNA from clones that overexpressed an inducible
UG band was prepared and the DNA sequence of the rhUG coding region
was again verified. Both plasmid DNA and single colony bacterial
isolates from streaks of positive clones were then frozen down for
storage at -20.degree. C. and -80.degree. C.
1TABLE 1 Oligonucleotides used in Construction of a Synthe- tic
Bacterial Gene rhUG Oli- go. ID Nucleotide Sequence 1
5'-GATCCATGGAAATCTGCCCGTCTTTCCAGC- GTGTTATCGAAAC
CCTGCTGATGGACACCCCGTCC-3 2
5'-AGCTACGAAGCAGCTATGGAACTGTTCTCTCCGGACCAGGA CATGCGTGAA
GCAGGTGCT-3' 3 5'-CAGCTGAAGAAACTGGTTGACACCCTGCCGCAGAAACCG- CGT G
AATCCATCATAAACTG-3' 4 5'-ATGGAGAAGATCGCTCAGTCTAGCCTGTGCAACTAAG-3' 5
5'-CTTAGTTGCACAGGCTAGACTGAGCGATCTTCTCCATCAGTTT G
ATGATGGATTCACGCG-3' 6 5'-GTTTCTGCGGCAGGGTGTCAACCAGTTTCTTC-
AGCTGAGCACT GCTTCACGCATGTCCT-3' 7
5'-GGTCCGGAGAGAACAGTTCCATAGCTGCTTCGTAGCTGGACG GGGTGTCCATCAGCAGGG-3'
8 5'-GGTCCGGAGAGAACAGTTCCATAGCTGCTT- CGTAGCTGGACG
GGGTGTCCATCAGCAGGG-3'
[0158] Oligonucleotides homologous to the 5' and 3' ends of the
synthetic gene, and containing flanking linkers with NcoI and BamHI
restriction sites, respectively, were then used to amplify the
synthetic bacterial gene by PCR and clone it into pKK223-3
(obtained from Pharmacia Corp.). The DNA sequence of the synthetic
gene in pKK233-3 was confirmed. Poor rhUG expression from this
clone suggested that extra amino acid residues would be required at
the N-terminus, in addition to the initiator methionine (Peter, et
al., 1989), in order to increase rhUG expression levels. Therefore,
a set of synthetic bacterial rhUG genes with N-terminal additions
of varying length were constructed to evaluate the optimal length
for rhUG expression in bacteria. These extra amino acids may help
to stabilize the nascent peptide in the bacterial ribosome and
cytoplasm. The extra amino acids consisted of alternating glycines
and serines, as these have small side chains, are not highly
charged or highly hydrophobic, and therefore are unlikely to
disrupt the natural folding and assembly of the UG monomers and
dimers. Codon usage and linkers were used as described above and
the genes were cloned into pKK223-3. The DNA sequence of each gene
was verified. Clones selected for correct rhUG coding sequence and
inducible expression of rhUG were inoculated from colonies on solid
media into 50 ml of broth and shaken overnight. This starter
culture is used to inoculate 250 ml of rich media containing
antibiotic in shaker flasks. These cultures were grown under the
appropriate conditions until they reached an optical density of 0.5
at 600 nm. Expression of rhUG was then induced for each clone (see
Table 2). The cultures were shaken for an additional 2-4 hours. The
cells were harvested by centrifugation, resuspended, and analyzed
for rhUG expression by SDS-PAGE. The rhUG expression levels from
each gene were compared and the gene that produced the most protein
was selected for further host/vector system optimization. The gene
producing the most protein had an N-terminus containing three extra
amino acid residues in addition to the human uteroglobin sequence
and is referred to as the MGS-gene.
[0159] There are significant disadvantages associated with the use
of pKK223-3. First, pKK223-3 is not suitable for production of a
biopharmaceutical in the United States because it requires
ampicillin selection for stable plasmid inheritance from parent to
daughter bacterial cells. Approximately 20% of the U.S. population
is allergic to penicillin and its derivatives, one of which is
ampicillin. For this reason, the FDA has barred the use of
ampicillin in processes used to generate recombinant
biopharmaceutical proteins. In the absence of ampicillin, the
plasmid can be lost from the cells in the culture as parent cells
divide and daughter cells containing the plasmid are not selected.
Plasmid DNA replication represents a significant metabolic burden
to the bacterial cells. In the absence of the antibiotic, the
daughter cells lacking the plasmid will have a competitive
advantage over daughter cells that still contain the plasmid and
will rapidly take over the culture.
[0160] Second, the transcription of the synthetic gene is repressed
by the lac repressor protein which binds to the lac promoter
element and prevents uninduced expression of the downstream
protein. Therefore, a high copy number of pKK223-3 may result in
more promoter elements than there are lac repressor proteins in the
cell, causing "leaky" protein expression. Transcription of the gene
downstream of the lac promoter is actually turned on by adding a
chemical (isopropyl thio-galactoside, "IPTG") that binds to the lac
repressor protein, causing it to let go of the lac promoter DNA.
Bacterial RNA polymerase then binds to the promoter and initiates
transcription. The de-repression of the promoter thus induces mRNA
transcription and protein expression. Leaky protein expression
occurs when the downstream protein, in this case rhUG, is
synthesized in the uninduced culture. Leaky rhUG expression from
pKK223-3 was observed.
EXAMPLE II
Testing of Plasmid Vector Constructs in Strains of E-Coli
[0161] Several different plasmid vector constructs containing the
MGS-synthetic gene and different combinations of replicons,
promoters, transcriptional repressors, and antibiotic selections
were then tested in several different strains of E. coli. Several
of these host/vector systems are shown in Table 2. A version of the
synthetic gene with MAA- at the N-terminus was also made and tested
in some of these host-vector systems. Subclonings of the synthetic
genes into pRK248cIts were done using a BamHI fragment containing
the rhUG synthetic gene from the pKK223-3 clones. Subclonings into
pGEL101 and pGELAC were done using NcoI-BamHI fragments containing
the gene in pKK223-3.
2TABLE 2 Combinations Generated for Optimal rhUG Expression in E.
coli Strain rhUG- ID N-terminus Vector 5'RE-3'RE Selection.sup.1
Induction Promoter Host Strain Source CG1 M- pKK223-3 Nco1-BamH1
Ampicillin.sup.2 IPTG.sup.5 Lac DH5.alpha.F'I.sup.q Life
Technologies Inc CG3 FLAG- pKK223-3 Nco1-BamH1 Ampicillin IPTG Lac
DH5.alpha.F'I.sup.q Life Tech CG4 MGS- pKK223-3 Nco1-BamH1
Ampicillin IPTG Lac DH5.alpha.F'Iq Life Tech CG5 MGSGS- pKK223-3
Nco1-BamH1 Ampicillin IPTG Lac DH5.alpha.F'Iq Life Tech CG8
MGSGSGS- pKK223-3 Nco1-BamH1 Ampicillin IPTG Lac DH5.alpha.F'Iq
Life Tech CG60 MGS- pGEL101 Nco1-BamH1 Ampicillin IPTG T7 RNA pol
BL21/DE3 Novagen, Inc. CG73 MGS- pRK248cIts-A.sup.7 BamH1-BamH1
Tetracycline.sup.3 Heat.sup.6 .lambda.PL DH5.alpha.F'Iq Life Tech
CG74 MGS- pRK248cIts-A BamH1-BamH1 Tetracycline IPTG T5/lacO hyb
DH5.alpha.F'Iq Life Tech CG77 MAA- pKK223-3 Nco1-BamH1 Ampicillin
IPTG T5/lacO hyb DH5.alpha.F'Iq Life Tech CG78 MGS- pRK248cIts-B
BamH1-BamH1 Tetracycline Heat .lambda.PL DH5.alpha.F'Iq Life Tech
CG82 MAA- pGELAC Nco1-BamH1 Ampicillin.sup.4 IPTG T5/lacO hyb
DH5.alpha.F'Iq Life Tech CG86 MGS- pRK248cIts-B BamH1-BamH1
Tetracycline IPTG T5/lacO hyb W3110F'Iq ATCC CG98 MAA- pGELAC
Nco1-BamH1 Ampicillin IPTG T5/lacO hyb DH1 ATCC Table 2 - Footnotes
.sup.1Antibiotic selection is required to select bacterial
transformants containing the plasmid but may not be necessary for
plasmid maintenance (e.g. stable inheritance of the plasmid).
.sup.2Ampicillin selection is done with 100 micrograms per
milliliter of solid or liquid culture media. .sup.3Tetracycline
selection for pRK248cIts is 20 micrograms per milliliter of solid
or liquid culture media. Tetracycline selection is not required for
stable inheritance of pRK248cIts under non-inducing conditions, but
is required when rhUG synthesis is induced. .sup.4Ampicillin
selection is not required for stable inheritance of pGELAC.
.sup.5The concentration of IPTG used to induce rhUG expression was
0.5 mM. .sup.6Transcription from the .lambda.P.sub.L is induced by
a rapid shift in the temperature of the culture from 32.degree. C.
to 42.degree. C. The temperature change causes a conformational
change in the lambda repressor protein, also expressed from the
pRK248cIts vector, that renders it unable to bind the
.lambda.P.sub.L promoter. The bacterial RNA polymerase is then able
to recognize the promoter and initiate transcription. .sup.7The "A"
signifies that the rhUG gene is in one orientation while the "B"
signifies that it is in the opposite orientation, in the same BamH1
site.
[0162] One significant advantage of using pRK248cIts is that it is
an oligo-copy number plasmid with a very stable origin of
replication derived from RP4. Most high copy number vectors allow
"leaky" protein expression in the uninduced state and are
inherently less stable than lower copy number plasmids. Although
pRK248cIts requires no selection for maintenance and stable
inheritance of the plasmid in daughter cells, it bears antibiotic
resistance genes for ampicillin and tetracycline. These antibiotics
can be used for the convenient selection of bacterial transformants
during clonings. However, plasmid stability is often impaired when
high level expression of a recombinant protein encoded on the
plasmid is induced. If an antibiotic is needed to maintain the
plasmid during expression of a recombinant protein, then
tetracycline could be used in biopharmaceutical production, which
is permitted by the FDA.
[0163] The synthetic gene was also inserted into pGEL101 (Mantile,
1993) and pGELAC (Mantile, 2000). The expression of rhUG from the
synthetic gene versus expression from the human cDNA sequence in
these plasmids was compared and the synthetic bacterial gene
yielded superior results.
[0164] The use of the par sequence (a 2.8 kb sequence derived from
the broad host range R factor RP4) to stabilize plasmid inheritance
has been described under chemostat conditions using ampicillin
selection after a prolonged period of growth in the absence of
antibiotic as the criterion for stability (Mantile, 2000). However,
the process of the invention does not involve the use of chemostat
conditions. Instead, the production strain (host/vector system) is
required to undergo a series of seed banking processes, involving
growth in the absence of ampicillin followed by a freeze-thaw
cycle. Then the host/vector system must remain stable in the
absence of ampicillin selection through a series of fermentations
to reach a large cell biomass before induction of protein
expression during which stable inheritance must be maintained so
that maximum protein levels are achieved.
[0165] Production strain CG12 is a host/vector system containing a
plasmid similar to pGELAC in E. coli strain BL21/DE3. This strain
was tested for both genetic stability (at the level of DNA
sequence) and plasmid stability in the absence of ampicillin
selection. The following examples demonstrate that the host/vector
system in production strain CG12 is stable in all respects
throughout the production process, from seed banking to final
harvest of the large scale induced fermentation culture.
Construction of a Separate Host-Vector System
[0166] The source of the UG protein coding sequence was cDNA
generated from human lung mRNA. The cGMP expression system for
recombinant human UG is similar to that described in Mantile et al.
(Mantile, 2000; Mantile, 1993, Miele, 1990). The protein is
expressed from a plasmid, called pCG12, using the T7 promoter and
the T7 DNA-dependent RNA polymerase, which is under control of the
IPTG-inducible lac promoter in the genome of the BL21/DE3 host
strain. The expression vector, pCG12, codes for a protein with
three additional amino acids at its N-terminus relative to the
native human protein (an initiator methionine followed by two
alanines). While these are necessary for efficient translation in
bacteria the methionine is cleaved off during the fermentation
process, resulting in a product comprising UG protein with two
additional alanines at the N-terminus.
[0167] The pCG12 expression vector is suitable for cGMP
manufacturing for several reasons. First, it lacks a requirement
for antibiotic selection during fermentation. Although pCG12 can be
selected using ampicillin, the antibiotic is not required to
maintain stable plasmid inheritance during bacterial cell growth
and propagation. Antibiotic-free stable plasmid inheritance was
achieved through the use of a plasmid stabilization sequence called
par. The par sequence is a 2.8 kilobase sequence that is derived
from the broad-host-range R factor, RP4 (Gerlitz, 1990). Par
modifies plasmid partitioning and significantly enhances plasmid
stability. This sequence confers long term stability under
chemostat conditions, for over 250 generations in the absence of
antibiotic selection (Mantile et al., 2000). Second, pCG12 is
genetically stable through the drug production process. Its DNA
sequence does not change, despite cell banking which involves
freezing and thawing steps that can break DNA. Third, it has been
shown the par sequence confers plasmid stability in the absence of
ampicillin selection, through the cell banking process,
subculturing, and in batch fermentations.
[0168] FIG. 3 shows the arrangement of genetic elements in pCG12.
This expression vector is similar to pGELAC (Mantile, 2000:
Genebank accession number HSU01102). The bacterial host strain for
expression of rhUG is BL21/DE3 (ATCC #47092). The 2.8 kilobase par
sequence is derived from the broad-host-range R factor, RP4, which
confers partitioning functions that enhance plasmid stability and,
it has been shown that it confers plasmid stability throughout the
production process, from cell banking to large scale
fermentation.
EXAMPLE III
Preparation of a Research Seed Cell Bank
[0169] A research seed culture was inoculated from a single colony
of BL21/DE3 containing pCG12 grown on LB agar containing 50
micrograms/ml of ampicillin. A research seed bank was generated
from the 50 ml research seed culture grown at 32.degree. C. in LB
medium containing no antibiotic selection. The culture was grown to
early stationary phase and glycerol was added to a final
concentration of 20%. The culture was then frozen in 1 ml aliquots
and stored at -75.degree. C. Aliquots of this research seed were
then used for fermentation development, as well as to generate
master and working cell banks.
[0170] The pCG12 vector is genetically stable, such that the DNA
sequence remains unchanged through the manipulations required to
produce rhUG drug substance. The entire pCG12 plasmid was sequenced
after cloning and prior to the creation of the research seed bank
(SEQ. ID NO. 9). Although pCG12 is stable in the absence of
antibiotic, it does confer ampicillin resistance upon its bacterial
host. The DNA sequence of the pCG12 plasmid recovered from
fermentation production cultures is identical to the plasmid
sequence from the research seed.
EXAMPLE IV
Preparation of Master and Production Seed Cell Banks
[0171] A master cell bank was prepared from research seed of strain
CG12. A flowchart outlining the both the Master and Production cell
banking processes is presented in FIG. 4. A list of the chemicals
and materials used in the manufacture of the Master and Production
seeds is provided in Table 3. All chemicals and materials were USP
grade, in compliance with cGMP.
3TABLE 3 Raw materials and Chemicals Used in Production of Master
and Production Seed Cell Banks. Material/Chemical Manufacturer
Grade Glycerol J. T. Baker USP/FCC Yeast Extract Difco N/A Tryptone
Difco N/A Sodium Chloride J. T. Baker USP/FCC Water for Injection
WRAIR USP Research Cell bank Claragen cGLP (for Master Seed
Production) Master Cell bank WRAIR cGMP (for Production Seed
Production)
[0172] An aliquot of the CG12 research seed was added to a shake
flask containing Luria Broth ("LB") and maintained at 32.degree. C.
with shaking, monitoring the growth by optical density (OD) from
550 nm to 660 nm, preferably at 600 nm. No antibiotic was used.
Samples of broth were subsequently taken from the shake flask at
approximately one hour intervals and the absorbance of each was
measured and recorded until an OD.sub.600 of 0.8 to 1.5 AU was
reached. One hundred milliliters of the culture were then combined
with 20 ml of the cryopreservative (glycerol) and a sample was
retained for Gram staining to verify the identity and purity of the
bacteria present in the culture. One milliliter of the culture was
transferred aseptically to each of 90 labeled cryovials, which were
placed into three labeled boxes each containing 30 vials. Two boxes
were transferred to a freezer maintained at -80.degree. C. and one
box was transferred to liquid nitrogen for storage.
[0173] The production cell bank was then prepared from the master
cell bank. The process for preparing the production cell bank was
identical to that used in preparing the master cell bank, except
that a vial from the master cell bank was used to start the culture
in place of the research seed. Ninety cryovials, each containing
one milliliter of the culture, were prepared and placed into three
boxes each containing 30 vials. Two boxes were transferred to a
freezer which was maintained at -80.degree. C. and one box was
transferred to liquid nitrogen for storage.
[0174] Each culture and cell bank was tested extensively and
results documented to comply with cGMP guidelines. The growth
curves of the master and production seed bank cultures showing the
absorbance at 600 nm as a function of time are shown in FIG. 5.
Both cultures reached logarithmic growth within a few hours and
were harvested in mid-logarithmic growth. Samples for initial
viability were taken at this time. Samples to test the viability of
each bank were taken one week after the seed vials were frozen.
Other tests and assays to qualify the banks for cGMP are described
below. The results of these assays for the Master and Production
seeds (Lots 0644 and 0645, respectively) are set forth in Tables 4
and 5, respectively. Both the Master and Production Seeds passed
all specifications. The loss of five to ten percent of the cell
viability was expected from the freezing of the cells.
[0175] The following assays were used in the characterization of
the Master seed cell bank, the Production seed cell bank and the
Fermentation.
[0176] Purity. LB plates were streaked using sterile techniques and
were incubated at 37.degree. C. Colonies were examined after 24 to
36 hours.
[0177] Viability. Cell viability was determined by plating serial
dilutions of the cell culture on LB agar plates with or without
ampicillin. Colonies were then counted and the results
recorded.
[0178] Gram Staining. A small amount of the culture to be tested
was transferred to a slide and the slide was allowed to air dry
before being heat fixed. The fixed cells were then stained with
crystal violet followed by Grams iodine. Cells were then examined
under an oil immersion lens at 1000.times.. Control organisms are
S. aureus and E. coli.
[0179] Colony Morphology. LB plates were streaked as described in
the SOP and incubated as described. Colonies were examined after 24
to 36 hours.
[0180] Colony Appearance. LB plates were streaked using sterile
technique and incubated at 37.degree. C. Colonies were examined
after 24 to 36 hours.
[0181] Optical Density. Cell density of the culture was determined
by the absorbance from 550 nm to 660 nm, preferentially at 600 nm
using a LKB spectrophotometer.
[0182] SDS-PAGE. Samples for SDS-PAGE for the fermentation as well
as for in process samples for the purification were run on 10-20%
Tricine gels (Novex). Samples were mixed 1:1 (v:v) with
2.times.Tricine SDS-PAGE loading buffer (Novex) and run until the
dye front was approximately 1 cm from the bottom of the gel. High
or low molecular weight range size markers (Amersham) were used as
standards. Gels were fixed by heating to at least 85.degree. C. for
5 minutes in the presence of 10% acetic acid/30% methanol followed
by staining with Gel Code Blue stain from Pierce Chemical Co.
Destaining was performed in purified water as described by Pierce.
Gels were then photographed and dried.
4TABLE 4 Assay Summary Table for Master Seed Lot No. 0644 Assay
Result Purity-Final Culture No Contamination Initial Viability of
Master Cell bank-LB 230 .times. 10.sup.6 CFU/ml Plates Initial
Viability of Master Cell bank-LB 420 .times. 10.sup.6 CFU/ml Plates
+ Ampicillin Gram Stain Gram (-) Rods without Contamination 1 Week
Post Manufacturing Viability of 85 .times. 10.sup.6 CFU/ml Master
Cell bank-LB Plates 1 Week Post Manufacturing Viability of 67
.times. 10.sup.6 CFU/ml Master Cell bank-LB + Ampicillin Plates
Colony Morphology-LB + Plates Creamy white single smooth colonies
Colony Morphology-LB + Ampicillin Creamy white single Plates smooth
colonies Colony Appearance-LB Plates Creamy White Colony
Appearance-LB + Ampicillin Creamy White Plates CFU = Colony Forming
Units
[0183]
5TABLE 5 Assay Summary Table for Production Seed Lot No. 0645 Assay
Result Purity-Final Culture No Contamination Initial Viability of
Production Cell bank- 270 .times. 10.sup.6 CFU/ml LB Plates Initial
Viability of Production Cell bank- 290 .times. 10.sup.6 CFU/ml LB
Plates + Ampicillin Gram Stain Gram (-) Rods without Contamination
1 Week Post Manufacturing Viability of 77 .times. 10.sup.6 CFU/ml
Production Cell bank-LB Plates 1 Week Post Manufacturing Viability
of 50 .times. 10.sup.6 CFU/ml Production Cell bank-LB + Ampicillin
Plates Colony Morphology-LB Plates Creamy white single smooth
colonies Colony Morphology-LB + Ampicillin Creamy white single
Plates smooth colonies Colony Appearance-LB Plates Creamy White
Colony Appearance-LB + Ampicillin Creamy White Plates CFU = Colony
Forming Units
[0184] The Production seed cell bank is used to inoculate
fermentations for production of rhUG and the Master seed cell bank
is used to create new Production seed cell banks as they are used
up. These two banks provide for a long-term qualified source of raw
material, e.g. bacterial cell paste, from which to purify
pharmaceutical grade rhUG.
EXAMPLE V
Fermentation
[0185] A list of the chemicals and equipment used in the
fermentation are provided in Tables 6 and 7, respectively.
6TABLE 6 Chemicals used in E. coli Fermentation for Production of
rhUG Chemical Manufacturer Grade Select APS Super Broth plus Difco
N/A Glycerol J. T. Baker USP/FCC
Isopropyl-.beta.-D-thiogalactopyranoside Sigma N/A Sodium Chloride
J. T. Baker USP/FCC Mazu DF 204 Mazer Chemical N/A
[0186]
7TABLE 7 Equipment used in E. coli Fermentation for the Production
of rhUG Equipment Manufacturer Model 400 L Fermenter System New
Brunswick Scientific IF-400 Biological Safety Cabinet Baker B60-ATS
Continuous Feed Centrifuge Sharples AS-Z6SP Shaker-Incubator New
Brunswick Scientific Innova 4330 pH meter Orion 420
Spectrophotometer LKB N/A Peristaltic pump Cole-Parmer 07523-40
Overhead Mixer Lightnin MSV-1500
[0187] A flowchart outlining the fermentation process is presented
in FIG. 6. To begin the fermentation process, a vial of the
Production seed cell bank was thawed at room temperature. One
hundred microliters of the production seed was then used to
inoculate each of the six, fernbach flasks containing one liter
each of sterile Super Broth medium (Becton-Dickinson Select APS
Super Broth, glycerol and WFI). The cultures in the six flasks were
then incubated at 32.degree. C. in a New Brunswick shaker-incubator
with agitation (300 rpm) for approximately 20 hours. The cultures
in the six flasks were then used to inoculate 300 liters of
Superbroth in a 400 liter New Brunswick Scientific Fermenter System
(Model IF-400).
[0188] Preparation of the fermenter prior to inoculation was as
follows: the fermenter was cleaned and sterilized according to
standard operating procedures, and was then charged with 100 kg of
WFI. Super Broth medium (Becton Dickinson Select APS Super Broth,
glycerol and WFI) was added to the fermenter and additional WFI was
added to reach a final, net weight of 300 kg. The fermenter was
then pressure tested and sterilized at 122.degree. C. for 30
minutes according to a standard operating procedure. Antifoam (Mazu
DF 204) was added to the fermentation as required. The set point
parameters for the fermentation are defined in Table 8.
8TABLE 8 Fermentation Set Point Parameters Parameter Set Point
Range Actual Set Point Agitation 150 .+-. 10 rpm 150 Temperature 32
.+-. 2 C 31.9 Air Flow 300 .+-. 2 L/min 299.8 L/min Pressure 3 .+-.
1 psig 2.4 psig Dissolved Oxygen NA .gtoreq.20%
[0189] The shake flask inoculum is then added to the fermenter and
the culture is grown at 25.degree. C. to 40.degree. C. until a
minimum optical density at 600 nm of 2.0 was reached. On reaching a
minimum OD.sub.600 of 2.0 the expression of rhUG is induced by the
addition of isopropyl-.beta.-D-thiogalactopyranoside (IPTG) to the
fermentation culture to a final concentration of 0.1 mM to 10 mM.
The fermentation was maintained for at least one hour, preferably
two hours post induction. The bacterial culture is harvested by
centrifugation with a Sharple's continuous feed centrifuge. The
cell paste is partitioned and stored frozen at -80.degree. C. for
later purification.
[0190] In the example shown, the six shake flask cultures used for
the inoculation of the fermenter reached an average OD.sub.600 of
2.8 after fourteen and a half hours and contained
210.times.10.sup.6 colony forming units per milliliter. During the
fermentation dissolved oxygen levels decreased in response to the
increased cell metabolism and biomass between three and four hours
into the fermentation. The agitation range was sufficient to
maintain the dissolved oxygen at a minimum level of 20%. Expression
of rhUG in the fermentation culture was induced after 4.2 hours
(with an OD.sub.600 of 2.7 and a cell count of 40.times.10.sup.6
CFU/ml). Growth continued at log phase rates for a little over an
hour post induction, cells were harvested after approximately 6
hours of fermentation (FIG. 7). Samples were taken from the culture
after fermentation and were analyzed later by SDS-PAGE (FIG. 8).
All other fermentation data is recorded in Table 9. The
fermentation passed all specifications.
9TABLE 9 Assay Results, E. coli Fermentation for the Production of
rhUG Lot No. 0708 Assay Result Sterility Check No Growth Gram Stain
Gram (-) Rods SDS-PAGE Comparable to Reference Final Viability on
LB 160 .times. 10.sup.6 CFU/ml Final Viability on LB- 110 .times.
10.sup.6 CFU/ml Ampicillin Purity of Final Samples No Contamination
Final Colony Morphology Creamy, Homogeneous, Noncontaminated,
Single colonies CFU = Colony Forming Units
EXAMPLE VI
Purification of rhUG
[0191] Chemicals, supplies and equipment which were used in the
purification of rhUG are shown in Tables 10 and 11.
10TABLE 10 Chemicals and Supplies Used in rhUG purification Process
Chemical/Supply Manufacturer Grade Ethanol, USP-200 Spectrum USP
Tris Base(Hydroxymethyl)-aminome- thane Spectrum USP/NF Sodium
phosphate, monobasic, J. T. Baker USP/NFF monohydrate Sodium
chloride J. T. Baker USP/NFF Sodium Hydroxide (pellets) Spectrum
NF/FCC Hydrochloric acid (concentrated) J. T. Baker USP/NFF
Edetate, disodium, dihydrate Spectrum USP/NF Copper sulfate,
pentahydrate J. T. Baker USP Macro Q 50 BioRad N/A Type I
Hydroxyapatite, 20.mu. Biorad N/A Chelating Sepharose Fast Flow
Pharmacia N/A 100 K Ultrafiltration cartridge Millipore N/A 5 K
Ultrafiltration cartridge(s) Millipore N/A Sartobind Q cartridge
Sartorious N/A Size 15, 24 and 73 Silicone tubing Sanitech N/A Size
73, Pharmed tubing Cole-Parmer N/A Size 191, Bioprene tubing Watson
Marlow N/A Millipak 20, 60, 100 and 200 sterile Millipore N/A
20.mu. filters E. coli Cell paste WRAIR cGMP SP Sepharose Fast Flow
Amersham N/A Phenyl Sepharose Fast Amersham N/A Flow/High
Substitution
[0192]
11TABLE 11 Instruments and Equipment Used in rhUG Purification
Process Item Supplier Model Number Pellicon 2 Cassette Filter
Millipore Pellicon 2 Stainless Steel Holder(s) Lab Masters SI mixer
Lightnin N/A Spectrophotometer Shimadzu UV 160 Vantage A column(s)
Amicon 18.0 .times. 50 cm Vantage A column Amicon 13.0 .times. 50
cm Balance Sartorius 14800p Variable Speed Peristaltic Pump, I/P
Millipore XX80EL0-00 Masterflex L/S pump(s) Cole-Parmer G-07523-20
Peristaltic Pump Watson Marlow 701 S/R Super Speed Centrifuge(s)
Sorvall RC-5B/RC-5C High Speed Rotor PTI 14C Fluidizer
Microfluidics M-110F 142 mm Stainless Steel holder Sartorius
16276-3 UV Monitor Pharmacia Uvicord SII chart Recorder Pharmacia
Rec I Conductivity Meter Orion 162 pH Meter System Orion 620
[0193] Batches of rhUG having common biological activities and
physical and chemical specifications were purified by minor
variations of the same process two of which followed cGMP
guidelines for pharmaceutical production. Two of the processes, one
of the cGMP purification processes and one process used for the
production of rhUG for animal studies, are outlined in FIG. 11.
Descriptions of these processes and of several variations used in
both cGMP processes and in the production of rhUG for animal
studies are as follows. For the cGMP process outlined in FIG. 11b,
one kilogram of bacterial cell paste was lysed by shear and the
cell debris removed by centrifugation. The lysate (supernatant) was
then processed using a 100 K nominal molecular weight cut off
(NMWCO) membrane in a tangential flow filtration (TFF) system. The
permeate from the 100 K step was concentrated by TFF using a 5 K
NMWCO membrane and loaded onto a Macro Q anion exchange column. The
eluate from the anion exchange column was concentrated and
diafiltered by TFF using a 5 K NMWCO membrane before being loaded
onto a Type I Hydroxyapatite (HA) column. The eluate from the HA
column was then loaded directly onto a column packed with Chelating
Sepharose Fast Flow (CSFF) resin with copper as the chelate. The
rhUG passed through the column while the host-derived proteins
present in the HA eluate bound to the column. A positively charged
Sartobind Q TFF membrane was also placed into the flowstream after
the copper CSFF column to ensure that the maximum amount of
endotoxin was removed from the final bulk material. The
pass-through from the Sartobind Q was concentrated and then
extensively diafiltered using a 5 K NMWCO membrane with saline for
injection (SFI) as the replacement buffer, both to remove residual
copper as well as to properly formulate the final bulk
material.
[0194] This process and minor variations thereof were used both for
a separate cGMP clinical lot as well as in lots used for animal
testing. These variations include: 1) use of either a 30 K NMWCO
membrane or a 50 K NMWCO membrane in place of the 100 K NMWCO
membrane for separation of rhUG from other proteins in clarified
the bacterial lysate; 2) filtration of the HA eluate through a 30 K
NMWCO TFF membrane rather than processing by Copper bound CSFF
column chromatography; and 3) removal of the SartoBind Q membrane
after the copper bound CSFF column chromatography. The final step
in the purification of a five to twenty volume diafiltration
against saline using a 5 K NMWCO membrane was used in all
cases.
[0195] These methods produced rhUG with comparable physical
characteristics and is sufficient to meet the FDA's cGMP
manufacturing requirements and requirements for use of rhUG in
animal studies. The rhUG preparations made by this process, and
minor variations thereof, are comparable in all respects: apparent
size, molecular weight, charge, N-terminal amino acid sequence,
amount of free thiol indicating correct formation of
cystine-cystine bonds, immunological recognition techniques such as
ELISA and Western blotting, and biological activity. Protein
purified using the copper CSFF column was tested for the presence
of copper by Inductively Coupled Plasma (by QTI Inc.). No copper
was detected and the detection limit of the assay was 0.5 ppm. This
translates into a maximal dose of 1 .mu.g per 2 ml dose, which is
well below the estimated safe and adequate daily dietary intake of
600 .mu.g per day for infants (Olivares, 1996).
[0196] Columns were packed using standard operating procedures and
according to the column and resin manufacturers' recommendations.
All packed columns were sanitized with 0.5 M sodium hydroxide for a
minimum of 30 minutes and placed into their respective storage
solutions until use. The membranes for the tangential flow
filtration were sanitized and depyrogenated with 0.5 M sodium
hydroxide at 45.+-.5.degree. C. for a minimum of one hour prior to
use. The Sartobind Q membranes were sanitized and depyrogenated
with 1.0 N NaOH for a minimum of 30 minutes prior to use.
[0197] Flowcharts showing the steps in embodiments of the
purification process are presented in FIGS. 10 through 16. One
kilogram of frozen cell paste from the fermentation was thawed at
room temperature and lysed by shear using either a
Microfluidizer.TM. (Microfluidics) or a similar shear device. The
resulting crude lysate was clarified by centrifugation at
15,000.times.g. The clarified cell lysate was purified by constant
volume diafiltration in 25 mM Tris/40 mM NaCl pH 7.0 using a 100 K
NMWCO membrane. The permeate from the 100 K TFF step was collected
and concentrated using a 5 K NMWCO membrane with. After
concentration of the 100 K permeate a 5.times.constant volume
diafiltration was performed with 25 mM Tris/40 mM NaCl pH 8.5 to
remove low molecular weight impurities and to change the 100 K TFF
buffer with Macro Q Anion exchange equilibration buffer to produce
the 5K Ret #1 (FIG. 10). This was then loaded onto a three liter
Macro Q anion exchange column. Non-bound and weakly bound proteins
were washed from the column and the fraction containing the rhUG
was eluted with 25 mM Tris, 150 mM sodium chloride, pH 8.5 (FIGS.
11a and 11b). The eluate from the Macro Q column was concentrated
and the buffer was simultaneously exchanged with the equilibration
buffer for the HA column using a 5 K NMWCO membrane to produce the
5K Ret. #2 (FIG. 12). The 5K Ret. #2 was loaded onto a three liter
Type I Ceramic Hydroxyapatite column. Non-bound proteins were
washed from the column with 10 mM Sodium Phosphate pH 7.0 and the
fraction containing the rhUG was eluted with 75 mM sodium
phosphate, pH 7.0 (FIGS. 13a and 13b). The eluate from the HA
column was loaded directly onto a one liter Chelating Sepharose
Fast Flow (CSFF) column charged with copper. The rhUG did not bind
to the copper CSFF column and was retrieved in the flowthrough
(FIGS. 14a and 14b). The flowthrough from the copper CSFF column
was then diluted one to one with WFI and passed through a Sartobind
Q filter as a final endotoxin removal step (FIG. 15). The
passthrough from the Sartobind Q membrane was concentrated using a
5 K NMWCO TFF membrane. After concentration, a 20.times.constant
volume diafiltration was performed with Saline for Injection as the
replacement buffer (FIG. 16). The diafiltered material was then
further concentrated to a minimum protein concentration of 7.5
mg/ml, filtered and diluted with Saline for Injection (SFI; 0.9%
NaCl) to a target concentration of 5.5 mg/ml. The rhUG was then
sterile filtered to generate the Purified rhUG Bulk Drug.
[0198] Two additional purification methods can be employed to
improve the quality of the rhUG biopharmaceutical product. The
first method involves further purifying rhUG by addition of a
cation exchange chromatography step. This column chromatography
method can be used at various points in the process to selectively
remove residual E. coli proteins, particularly from the final
product. For example, in FIG. 17, rhUG (also known as rhCC1O) is
purified on an SPSFF column using a step gradient of NaCl. The
buffer is Sodium acetate pH 4.0. The rhUG preparation from the
Hydroxyapatite column was adjusted to a pH of 4.0 and run through a
SP Sepharose Fast Flow (SPSFF) cation exchange column (Amersham.)
RhUG alone eluted from the column with a sodium chloride step
gradient as shown in FIG. 17, while E. coli proteins eluted earlier
and later. Therefore, addition of a cation exchange step, or
substitution of cation exchange for hydroxyapatite chromatography
or other chromatography steps, can significantly improve the purity
of the final rhUG biopharmaceutical product.
[0199] The second method for improvement of purification involves
the use of hydrophobic interaction chromatography (HIC) to remove
residual E. coli proteins and residual and other misfolded forms of
the protein rhUG. For example, rhUG material from the Copper IMAC
(Cu-IMAC) column is loaded onto a Phenyl Sepharose Fast Flow/High
Substitution (PSFF/HS) column from Amersham. Ammonium sulfate is
added to the rhUG preparation from the Copper IMAC column to
increase specific binding of rhUG to the PSFF/HS column. Desirable
rhUG, the anti-parallel homodimer, passes through the column and is
recovered in the flow-through. Residual E. coli proteins, rhUG
aggregates and other misfolded or misaligned forms of rhUG are
eluted from the column by a decrease in the ammonium sulfate in the
mobile phase as shown in FIG. 18. In this example, anti-parallel
rhUG, also known as rhCC10, is purified on a PSFF/HS column using a
step gradient of ammonium sulfate. The buffer is Sodium phosphate
pH 7.0. Analysis of the rhUG from the Cu-IMAC column indicated the
presence of homodimers in the parallel orientation (an undesireable
contaminant). These data are shown in Table 12, which shows an
LC/MS analysis of peptides generated from digestion of rhUG
preparations with Endoproteonase Lys-C. Peptides derived from both
parallel and anti-parallel orientations are present in rhUG
preparations that have not been processed with an HIC step,
although the anti-parallel form is the predominant form (>90%).
Therefore, addition of a hydrophobic interaction chromatography, or
substitution of HIC for another step in the purification process,
can significantly improve the homogeneity of the anti-parallel rhUG
biopharmaceutical product.
[0200] In Table 12, peptide fragments were obtained by digestion of
the rhUG by Endoproteonase Lys-C which cleaves after Lysine
residues. The peptides were then separated using RP-HPLC and
fragments were analyzed both by absorbance at 214 nm and by
analysis by positive ion-electrospray mass spectrometry. The
material below was obtained from a sample of Cu-IMAC flow-through
material.
12TABLE 12 Fragments Obtained by Digestion of rhUG by
Endoproteonase Lys-C Expected Fragments Mass Type A(1)-K(44)
4888.307 Monomer K(45)-K(45) 146.105 Monomer L(46)-K(53) 912.528
Monomer P(54)-K(60) 841.502 Monomer L(61)-K(64) 519.273 Monomer
I(65)-N(720 834.390 Monomer A(1)-K(44)-S-S-A(1)-K(44) 9776.1 Dimer
(Parallel) A(1)-K(44)-S-S-I(65)-N(72) 5722.70 Dimer (Anti-Parallel)
I(65)-N(72)-S-S-I(65)-N(72) 1668.78 Dimer (Parallel)
A(1)-K(45)-S-S-A(1)-K(44) 9922.72 Dimer (Parallel)
A(1)-K(45)-S-S-I(65)-N(72) 5868.80 Dimer (Anti-Parallel)
A(1)-K(45)-S-S-A(1)-K(45) 10068.82 Dimer (Parallel)
[0201] The level of parallel dimer was either reduced or eliminated
in the material which passed through the PSFF/HS column indicating
full or partial removal of the misaligned parallel form from the
anti-parallel form desired in the biopharmaceutical formulation.
These results were repeated with several other types of HIC media,
in addition to phenyl Sepharose with similar results. Use of this
type of column provides a novel and unexpected means for the
separation previously unknown forms of rhUG.
[0202] The following assays were established as in process assays,
characterization assays and release assays for the production
process and for the drug substance and drug product. The rhUG drug
substances and drug products were compared to standard research lot
rhUG/7 where appropriate.
[0203] Western Blot. Two Western blots were performed, one with
a-rhUG antibody and one with .alpha.-E. coli lysate antibody (both
from Dako, USA). The .alpha.-rhUG Western was performed using a
rabbit polyclonal antibody to human UG with goat .alpha.-rabbit
IgG-HRP conjugate from DAKO as the secondary antibody. The
.alpha.-E. coli Western was performed with rabbit .alpha.-E. coli
lysate polyclonal antibody followed by a goat .alpha.-rabbit
IgG-HRP conjugate as the secondary antibody, both antibodies for
the .alpha.-E. coli assay were obtained from DAKO. Detection was
performed using the ECL.TM. kit from Amersham.
[0204] Bacterial Nucleic Acids. Bacterial DNA content per dose of
the rhUG drug substance and drug product was determined by Southern
blot using radiolabeled bacterial DNA followed by hybridization to
blotted concentrated rhUG sample (Charles River
Laboratories-Malvern).
[0205] Mass Spectroscopy. The molecular weight was determined by
Electrospray Ionization spectrometry by M-Scan Inc. Theoretical
molecular weight was determined by PAWS (a shareware program for
the determination of average molecular mass, obtained through Swiss
Pro). A value of 16110.6 Da was determined by the PAWS program. The
same value was found for cGMP batches of rhUG and was confirmed by
MS analysis of standard research lot rhUG/7 as a control
(determined molecular weight 16110.6 Da).
[0206] N-terminal Sequence analysis. The sequence of the N-terminus
was carried out using pulsed phase N-terminal sequencing on an
Applied Biosystems (ABI) 477A automatic protein sequencer. The
analysis was performed by M-Scan Inc. A sequence of Ala-Ala-Glu-Ile
was confirmed for cGMP batches of rhUG with standard research lot
rhUG/7 as a control.
[0207] pH. A three-point calibration (4.0, 7.0, 10.0) is performed
according to the manufacturers' instructions. After calibration of
the electrode the pH of the sample is determined.
[0208] Isoelectric Focusing. The pI was determined by isoelectric
focusing using gels with a pH range of 3 to 7. The gels were
obtained from Novex and were run under conditions as described by
the manufacturer. Samples were run versus a standard from Sigma and
a rhUG control (research lot rhUG/7). Gels were fixed by heating in
a microwave for 1 minute in the presence of 10% acetic acid/30%
methanol followed by staining with Gel Code Blue stain from Pierce.
Destaining was performed in purified water as described by
Pierce.
[0209] Free Thiol. The presence of free thiol was determined by
reaction with Ellman's reagent from Pierce using a modified
proticol to increase sensitivity. After incubation in the presence
of Ellman's reagent the absorbance of samples was determined in the
spectrophotometer at 412 nm. An extinction coefficient of 14150
M.sup.-1 cm-.sup.1 was used to determine the molar amount of free
thiol. A standard curve of free thiol (cysteine) was used to
monitor the linearity of the reaction.
[0210] LAL. The presence of bacterial endotoxin in rhUG process
intermediates, drug substance and drug product was tested by the
Limulus ameobocyte lysate assay as described in United States
Pharmacopeia (USP) Assay No. 85. Kits were obtained from Associates
of Cape Cod.
[0211] Color, Appearance, Homogeneity. The bulk drug product was
visually inspected for clarity, color and visible particulate
matter.
[0212] Immunoreactivity. A competitive ELISA was performed using an
antibody raised to native human UG isolated from urine (DAKO,
.alpha.-urine protein-1) as the capture reagent and a rhUG-HRP
(horseradish peroxidase) conjugate to compete with the rhUG in the
sample. The antibody was coated at a dilution of 2,500 onto
microtiter wells (100 microliters/well) in a 0.1 M
carbonate/bicarbonate buffer at pH 9.5 overnight. The wells were
dried and stored at 4.degree. C. until use. The rhUG-HRP conjugate
was made using a kit from Pierce. Approximately 250 nanograms of
the rhUG-HRP conjugate in 250 microliters of phosphate-buffered
saline (PBS) was used per well. A standard curve for each set of
samples was run using rhUG calibrators (research lot rhUG/7),
ranging from 0-500 nanograms/ml (shown in FIG. 19). All calibrators
and test samples were run in duplicate. The UG in the sample
competes with the rhUG-HRP conjugate for antibody binding sites in
the wells. Thus, the assay signal decreases with increasing amounts
of UG in the sample. The results were visualized by the
o-phenyldiamine dihydrochloride (OPD) HRP assay by Pierce. Plates
were read at 490 nm using a Biotek EL-80 microplate reader and the
data were analyzed using Biotek KC4 software.
[0213] Purity and Identity: Reducing SDS PAGE. The rhUG drug
substance and drug product was run on a Novex 10-20% Tricine
SDS-PAGE gel under both reducing and non-reducing conditions as
described by the manufacturer. Low molecular weight size standards
were obtained from Amersham. Gels were fixed by heating in a
microwave for 1 minute in a mixture of 10% acetic acid/30% methanol
and stained with brilliant blue R250 (0.5%, w/v). Gels were
destained with Novex Gel-Clear destaining solution as described by
the manufacturer. Gels were then dried using the Novex Gel-Dry
system and the percent purity was determined by scanning the gel
(Hewlett-Packard scanner Model 5100C) and densitometry was
performed using Scion Image shareware from the NIH.
[0214] Aggregation Assay. The drug product was analyzed for the
presence of aggregates by chromatography on either a Superose 12 or
a Sephadex 75 size exclusion chromatography (SEC) column
(Amersham/Pharmacia). The column was run according to the
manufacturer's instructions using the BioRad Biologic system and
peak area was determined using EZLogic Chromatography Analysis
software, also from BioRad. The percent aggregation was determined
by comparing the total area of all peaks vs. the area of peaks
eluting prior to the main UG peak.
[0215] Endotoxin. Endotoxin levels were tested by the rabbit
pyrogenicity assay as described in the U.S. Pat. No. 151. An amount
of rhUG equivalent to a single human dose was administered
intravenously over a 10 minute period. Body temperature increase
relative to the baseline pre-dose temperature was monitored over
the course of three hours. Acceptable results consist of no
temperature rise equal to or greater than 0.5.degree. C. over the
baseline results.
[0216] Protein Content. The protein contents of the process
intermediates, drug substance and product were determined by the
absorbance at 280 nm using a Shimadzu 120 and an extinction
coefficient of 2070 M.sup.-1 cm.sup.-1 as determined by Mantile et
al. (Mantile, 1993).
[0217] Sterility. The sterility assay was performed as described in
the U.S. Pat. No. 71. Samples were incubated into Fluid
Thioglycolate Media (FTM) and Tripticase Soy Broth (TSB). Positive
controls for TSB media were C. albicans, A. niger, and B. subtilis.
Positive controls for FTM were S. aureus, P. aeruginusa, C.
sporogenes.
[0218] Potency Assays. There are several biological activities
attributed to UG throughout the literature on the human protein and
its mammalian homologues. The biological activities that are
associated with preparations of human rhUG that are prepared
according to the described production process are described herein
and in U.S. Ser. Nos. 08/864,357, 09/087,210, 09/120,264, and
09/549,926.
[0219] Certain biological activities of UG can be measured in vitro
with available reagents and are relevant to the treatment of
certain diseases. Two of these activities have been verified for UG
as described herein. The first is an anti-inflammatory effect
arising from inhibition or blocking of secretory phospholipase
A.sub.2 enzymes (sPLA.sub.2s) by rhUG. We have confirmed that rhUG
significantly inhibits human sPLA.sub.2 enzymes in vitro,
specifically the Type Ib enzyme found in the pancreas and lung, as
well as the Type IIa enzyme produced by macrophages and found in
human rheumatoid synovial fluid. A novel fluorescence-based HPLC
assay for the inhibition of sPLA.sub.2-Ib activity by rhUG was
developed and has been used in conjunction with a more standard
.sup.14C-labeled assay. A second biological activity for rhUG is
its ability to bind to fibronectin which prevents inappropriate
deposition and the subsequent formation of a pro-fibrotic
extracellular matrix in a transgenic knockout mouse model of UG
deficiency (Zhang, 1997). A novel in vitro ELISA-type assay using
human fibronectin to measure this activity of rhUG was
developed.
[0220] These two potency assays can be used to gauge the relative
strengths of the in vivo biological activities of future batches of
rhUG. Because production processes, whether chemical or biological,
are inherently variable, potency assays are essential in assessing
potential safety and efficacy. The relative strength of the
biological activity may be determined by the potency assays.
[0221] Inhibition of secretory PLA.sub.2--Type Ib
[0222] The potency assay is based on the inhibition of rhPLA.sub.2
activity by the addition of rhCC10. RhPLA.sub.2 catalyzes cleavage
of the ester at the 2 position of L-3-phosphatidylcholine. Two
different assays were employed to measure this activity; one uses
as a substrate, 1-stearoyl-2-[1-.sup.14C]arachidonyl phosphotidyl
choline (Amersham) to produce [1-.sup.14C]arachidonic acid
(Product), which was then separated by liquid-liquid separation and
and the level of cleavage determined by scintillation counting
(PLA.sub.2 Assay No. 1). The second was performed using a
fluorescently labeled substrate, 2-decanoyl-1-(O-(11-(4,4-difluor-
o-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-
-glycero-3-phosphocholine (Molecular Probes). As shown in FIG. 18,
the uncleaved substrate was separated from the cleaved substrate
using normal phase HPLC, quantitation was performed using an in
line fluorescence detector (PLA.sub.2 Assay No. 2). Both assays
were performed in Hanks Balance Salt Solution with 1 mM CaCl.sub.2
in a final assay volume of 100 .mu.L. RhPLA.sub.2 was obtained from
Dr. Won Hwa Cho's laboratory at the University of Illinois-Chicago.
The novel PLA.sub.2 Assay No. 1 assay was performed as follows.
RhPLA.sub.2 is added to all tubes to a final concentration of 200
nM. For inhibition assays rhCC10 was added to a final concentration
of approximately 34 uM, an equal volume of HBSS was added to the
control tubes. All tubes were preincubated for 20 minutes at
37.degree. C., the assay was initiated by the addition of the
radiolabeled phosphotidylcholine to a final concentration of 5
.mu.g/ml. The reaction was then stopped after 15 minutes by the
addition of a 7.7 fold dilution of Doles reagent and purified water
(84:16). All tubes were vortexed and then centrifuged to separate
the hydrophobic and the hydrophilic layers. The top layer was then
removed and added to an Eppendorf tube containing 15 mg of silica
gel, mesh size 60 to 200, and 800 .mu.l of hexane for scintillation
counting.
[0223] The novel PLA.sub.2 Assay No. 2 was performed as follows.
RhPLA.sub.2 was added to all tubes to a final concentration of 200
nM. For inhibition assays rhCC10 was added to a final concentration
of approximately 34 uM, an equal volume of saline was added to the
control tubes. All tubes were preincubated for 20 minutes at
37.degree. C., the assay was initiated by the addition of
fluorescent labeled phosphotidylcholine to a final concentration of
5 .mu.g/ml. The reaction was stopped after 15 minutes by the
addition of a one to five dilution of 2-Propanol:n-hexane (8:3).
One hundred .mu.l of the stopped assay was loaded directly onto a
silica normal phase HPLC column. The mobile phase for the system is
2-propanol:n-Hexane:water (8:3:2). The amount of cleaved substrate
was determined by fluorescence with excitation at 480 rim and
emmision at 517 nm.
[0224] The percent inhibition for each assay was defined as:
% Inhibition=(1-(Substrate cleaved in the presence of
rhCC10.div.substrate cleaved in the absence of
rhCC10)).times.100.
13TABLE 13 Inhibition of sPLA2 by Different Lots of rhUG RhUG Lot
Numbers Assay CC10/6 CC10/7 CC10/8 0728 PLA.sub.2 Inhibition Assay
# 1: 48% 38% 54% 58% Radiolabel Assay PLA.sub.2 Inhibition Assay #
2: 59% 56% 69% 76% Fluorescence Assay
[0225] Binding to Human Fibronectin
[0226] A second biological activity for rhCC10 is its ability to
bind to fibronectin, which prevents inappropriate deposition and
the subsequent formation of a pro-fibrotic extracellular matrix in
a transgenic knockout mouse model of CC10 deficiency (Zhang, 1997).
A novel in vitro ELISA-type assay was developed using a recombinant
7 kDa fragment of human fibronectin (Fn III.1, also known as
fragment-III.sub.1-C, referred to as "rhFn") to measure this
activity and this assay was used to monitor biological activity of
rhCC10. Microtiter plates were coated with the fibronectin fragment
overnight and binding of rhCC10 was detected by competition with a
rhCC10-HRP (horse radish peroxidase) conjugate. RhCC10-HRP
conjugate was added to the plates and allowed to incubate for 1
hour at room temperature. The conjugate may be added with or
without standard or sample. PBS was used as a negative control. The
plate was aspirated and washed four times. The assay was visualized
by the o-phenyldiamine dihydrochloride (OPD) HRP assay from Pierce.
The plate was read at 490 nm using a Biotek EL-80 microplate reader
and the data was analyzed using Biotek KC4 software. FIG. 21 shows
a typical standard curve for this assay. The results of this assay
for all research and cGMP lots of rhUG were positive for binding of
rhUG to the fibronectin fragment.
[0227] In addition to the extensive testing and characterization of
the drug substance and drug products, samples of intermediates were
taken throughout the process to follow the purification and
determine the efficiency of each step. The process intermediates
were analyzed by SDS-PAGE, rhUG ELISA, LAL and for protein content.
Protein content was determined with a BCA assay from Pierce using
bovine serum albumin as a standard. All buffers were analyzed for
endotoxin content by the LAL assay. No endotoxin was detected in
the buffers.
[0228] The purification process was analyzed for both overall and
step recovery (Table 14). Overall purification, as determined from
the 100 K Bulk, was 51.9 percent. Purity as described by Specific
Activity was also examined (Table 15). Specific Activity is defined
as the value for the UG ELISA divided by the value for the BCA
protein assay for a defined sample. The largest amount of
impurities removed occurs in the 100 K diafiltration step and in
the subsequent Macro Q, anion exchange step. This is confirmed by
the data from the SDS-PAGE results (FIGS. 22a and 22b). The
Hydroxyapatite and Copper Chelating Sepharose Fast Flow columns are
required to remove the final E. Coli protein impurities. Recoveries
which exceed 100 percent and specific activities which exceed 1.00
are due to variability within the assays and to the different
standards used.
[0229] Endotoxin levels were followed throughout the purification
by the LAL assay. Endotoxin was 2400 EU/ml (total amount was
11.times.10.sup.6 EU) in the 5K Retentate #1. After the material
had been further purified on the Macro Q column, the endotoxin
concentration had fallen to 0.17 EU/ml in the 5K Retentate #2
(volume=4000 ml) for a total of 680 EU. This represents a
16,000-fold decrease in the endotoxin level. Endotoxin levels were
undetectable throughout the remainder of the purification.
14TABLE 14 Recovery of RhUG from Purification Lot 0726 Total
Overall Step Volume RhUG rhUG Recov- Recovery Step (ml) (mg/ml)
(mg) ery (%) (%).sup.1 Supernatant lysed 3990 6.45 25700 100 N/A
Cells 5K Ret #1 4780 4.67 22300 86.7 86.7 Macro Q Eluate 10000 2.10
21000 81.6 94.1 Hydroxyapatite Eluate 4000 4.37 17500 67.9 83.2
Chelating Sepharose 4250 3.57 15200 58.9 86.8 Pass Through
Sartobind Q Pass 9200 2.20 20200 78.6 134 Through Purified rhUG
Bulk 2474 5.40 13400 51.9 66.0 .sup.1Step Recovery is defined as
the recovery for each step.
[0230]
15TABLE 15 Specific Activity of rhUG from lot 0726 rhUG Protein
Specific Step (mg/ml) (mg/ml) Activity Supernatant lysed Cells 6.45
22.1 0.292 5 K Ret. #1 4.67 4.94 0.945 Macro Q Eluate 2.10 1.19
1.77 Hydroxyapatite Eluate 4.37 2.41 1.81 Chelating Sepharose Pass
Through 3.57 2.23 1.60 Sartobind Q Pass Through 2.20 0.89 2.48
Purified rhUG Bulk 5.40 3.08 1.75
[0231] The final, sterile filtered bulk Drug Substance passed all
criteria as shown in Table 16.
16TABLE 16 Specifications and Results for rhUG Drug Substance Lot
0726 Test Specification Results Color Clear, colorless Clear,
colorless Appearance No turbidity No turbidity Homogeneity
Homogeneous Homogeneous Immunoreactivity Positive reaction Positive
reaction Purity .gtoreq.95% 98.3% Aggregation .ltoreq.5% 0.18%
Endotoxin by Rabbit Satisfactory Satisfactory pyrogenicity Protein
content 5.5 .+-. 0.5 mg/ml 5.5 mg/ml Sterility Sterile Sterile
Biological activity Positive Positive Western blot .alpha.-rhUG
Consistent with rhUG Consistent with rhUG results from SDS-PAGE
results from SDS-PAGE .alpha.-E. coli One light band at .about.40 k
One light band at .about.40 kD Bacterial nucleic <100 pg/dose
<7.5 pg DNA/dose acids Mass spectroscopy App. 16110 16111.9 kDa
PH 5-8 6.30 Isoelectric focusing App. 4.7 4.7 Free Thiol <10%
(w/w) Not detectable. LAL <5 EU/mg <0.01 EU/mg N-terminal
Sequencing A-A-E-I A-A-E-I1 .sup.1Both MAAEI and AEI forms were
less than 0.062% of the total.
[0232] Due to the structure of rhUG both the dimer and the monomer
run at a lower molecular weight on SDS-PAGE than would be predicted
by the sequence molecular weight (FIG. 23). Another characteristic
of the protein is that separation of the dimer into monomers in the
presence of reducing agents is not complete, as can be seen by the
presence of residual dimer in lanes 5 and 9 of the
Coomassie-stained SDS-PAGE gel (FIG. 23) and in lane 5 of the
.alpha.-UG Western (FIG. 24). While rhUG is apparent at both the
dimer and monomer positions of lane 5 of the .alpha.-rhUG Western
(FIG. 24), there was no E. coli protein detectable in either the
monomer or the dimer position in lane 3 of the .alpha.-E. coli
Western (FIG. 25). The only visible band in lane 3 of the
.alpha.-E. coli Western has an apparent molecular weight of
approximately 40 kD (FIG. 25).
[0233] Another characteristic of rhUG is the formation of a small
quantity of aggregates, as is apparent in lane 11 of the
Coomassie-stained gel (FIG. 23) where the higher molecular weight
bands corresponded well with higher molecular weight bands in lane
2 of the .alpha.-rhUG western (FIG. 24). Both the dimer and the
aggregates appear to react more strongly with the .alpha.-rhUG
antibody than the monomer, consistent with observations made during
the development of the UG ELISA. Analysis of aggregates at 214 nm
by size exclusion chromatography indicates minimal formation of
rhUG aggregates as compared to the overall amount of dimer (Table
16).
[0234] The isoelectric point for rhUG was determined to be 4.7
using an IEF gel (FIG. 26). The results were confirmed by
submission of the amino acid sequence to Swiss Pro (www.expasy.ch),
the calculated pI (4.7) was the same as the observed pI.
EXAMPLE VII
Stability of Drug Substance
[0235] The exemplary Drug Substance and the exemplary Drug Product
are at the same concentration and in the same formula (i.e. no
excipients are added). Stability was tested on the Drug
Product.
[0236] Formulation and Packaging of the Drug Product
[0237] All materials, chemicals and equipment used in the final
fill of the exemplary Drug Product are listed in Tables 17 through
19.
17TABLE 17 Materials used in the Final Fill of the Drug Product
Item Manufacturer 2 ml vials Wheaton V-35 13 mm Stoppers West 13 mm
Aluminum Crimp Sealers Wheaton 2 mm Tubing Assembly Wheaton Forceps
- 6" N/A Aluminum Foil N/A 600 ml Beaker Kimax
[0238]
18TABLE 18 Chemicals used in the Final Fill of the Drug Product
Chemical Manufacturer Bulk rhUG WRAIR Sterile 70% Isopropanol
Veltrek
[0239]
19TABLE 19 Equipment used in the Final Fill of the Drug Product
Equipment Manufacturer Balance Sartorius Omnispense Wheaton Crimper
Kebby
[0240] The composition of an exemplary embodiment of the Drug
product is: rhUG at 5.5 mg/ml, and sodium chloride at 0.9% (w/v).
All filling operations were performed in a Class 100 environment
room. Both the room and the operations for fill were validated by
the operator. An Omnispense pump was set up with 2 mm tubing,
primed and set to fill to a weight of 2.0 g.+-.5% (1.90-2.10 ml). A
flowchart outlining the fill processes is shown in FIG. 27. A two
ml vial was tared and bulk rhUG was dispensed into the vial. After
filling, the weight of the vial was recorded. This procedure was
repeated two times. If the fill weights of the three vials were all
within the specified range, then all of the vials were filled. If a
vial fell out of the specified range, the dispenser volume was
adjusted and the process was repeated. After filling, vials were
stoppered manually and aluminum crimp seals were placed onto the
vials. The vials were crimped using a Kebby Power Crimp. Vials were
then labeled and inspected visually. The rhUG drug product produced
in this manner provides a clear, colorless solution with no visible
particulates.
[0241] Summary of Physical and Chemical Characteristics of the Drug
Product
[0242] RhUG is a dimeric protein with a molecular weight of 16110
kilodaltons as calculated from the amino acid sequence and
confirmed by electrospray mass spectroscopy. The protein is
composed of two subunits bound to one another by two cystine bonds.
Relative subunit molecular weight and the presence of the cystine
bonds has been determined by SDS-PAGE under reducing and
non-reducing conditions. The DNA sequence of the bacterial strain,
CG12, was confirmed as was the amino acid sequence of the
N-terminus of the protein by Edman degradation. The sequence of the
N-terminus was Ala-Ala-Glu-Ile as predicted (SEQ. ID NO. 10).
Cysteine is not readily detected by this method both due to the
inherent chemistry and to the fact that the cysteine is involved in
sulfur bonding.
[0243] The final, vialed rhUG drug product passed all specification
as shown in Table 20.
20TABLE 20 Specifications for rhUG Drug Product Lot 0728 Test
Specification Results Color Clear, colorless Clear, colorless
Appearance No turbidity No turbidity Homogeneity Homogeneous
Homogeneous Purity .gtoreq.95% 97.4% Aggregation .ltoreq.5% 2.25%
Endotoxin Satisfactory Satisfactory Protein content 5.5 .+-. 0.5
mg/ml 5.5 mg/ml Sterility Sterile Sterile Biological activity
Positive Positive Western blot .alpha.-rhUG Consistent with rhUG
Consistent with rhUG results from SDS-PAGE results from SDS-PAGE
.alpha.-E. coli One light band at .about.40 k One light band at
.about.40 kD Bacterial nucleic acids <100 pg/dose <1.6 pg DNA
per mg rhUG Mass spectroscopy App. 16110 16112.6 pH 5-8 6.82
Isoelectric focusing App. 4.7 4.7 Free Thiol <10% (w/w) Not
Detected LAL <5 EU/mg <0.01 EU/mg N-terminal Sequencing
A-A-E-I A-A-E-I.sup.1 Copper <16 .mu.M <16 .mu.M .sup.1Both
MAAEI and AEI forms were less than 0.062% of the total.
[0244] As was described for the Drug Substance, both the dimer and
the monomer of the Drug Product run to a lower molecular weight on
SDS-PAGE than would be predicted by the sequence molecular weight
(FIG. 28). Separation of the dimer into monomers of the Drug
Product in the presence of reducing agents was not complete as
demonstrated by the presence of residual dimer in lanes 5, 9, and
11 of the Coomassie gel (FIG. 28) and in lane 6 of the a-rhUG
Western (FIG. 29). While rhUG is apparent at both the dimer and
monomer positions of lane 6 of the .alpha.-rhUG Western (FIG. 29),
there was no E. coli protein detected in either the monomer or the
dimer position in lane 4 of the .alpha.-E. coli Western (FIG. 30).
There were no bands visible in lane 4 of the .alpha.-E. coli
Western (FIG. 30).
[0245] Aggregates were also apparent in lane 3 of the .alpha.-rhUG
Western (FIG. 29). Both the dimer and the aggregates appear to
react more strongly with the .alpha.-UG antibody than the monomer;
this was also observed in the development of the UG ELISA. Analysis
of aggregates at 214 nm by size exclusion chromatography indicates
minimal formation of rhUG aggregates as compared to the overall
amount of dimer (Table 20).
[0246] The isoelectric point for rhUG was determined to be 4.7
using an IEF gel (FIG. 31). The results were confirmed by
submission of the amino acid sequence to Swiss Pro, the calculated
pI was the same as the observed pI.
[0247] All pre-clinical development lots made for animal testing
were analyzed using similar techniques as used for the cGMP lots.
Ranges for critical parameters for the rhCC10 are presented in
Table 21. Other critical parameters such as pI, molecular weight,
N-terminal end sequence and free thiol were essentially identical
for all lots of rhUG.
21TABLE 21 Ranges for development lots rhCC10/6, rhCC10/7,
rhCC10/8, and cGMP lots 0728 and 0853. Assay Range of results
Purity 97.4% to >99.5% Aggregation 0.13% to 3.4% PLA.sub.2
Inhibition (Radioactive assay) 37.5% to 57.7% PLA.sub.2 Inhibition
(Fluorescent assay) 56.0% to 86.0%
[0248] The final Drug Product passed all release criteria and was
identical to the material used in the animal studies and would be
acceptable for use in a Phase I/II human clinical trial by the U.S.
FDA.
[0249] Stability of rhUG Preparations
[0250] Long term stability studies on purified rhUG preparations, a
developmental lot (GLP material; lot number rhUG/7 stored at
2-8.degree. C.) and a pharmaceutical grade manufacturing lot (drug
product lot number 0728 stored at 2-8.degree. C.), were carried out
for 18 and 15 months, respectively and for 7 months for accelerated
aging of a pharmaceutical grade manufacturing lot (drug product lot
number 0728 stored at 25.degree. C. and 60% Relative Humidity). At
specified times a vial of each was removed from storage at
2-8.degree. C. and tested. Assays are described in Table 22.
22TABLE 22 Assay performed for Stability Assessments 3Test
Specification Purity (Reduced SDS PAGE) .gtoreq.95% Aggregation
.ltoreq.5% Biological activity Positive Isoelectric focusing App.
4.7 Free Thiol <10%
[0251] Results for the assays for the research lot are presented in
Table 23 and assay results for the cGMP lot are shown in Table 24
(2-8.degree. C.) and Table 25 (25.degree. C. and 60% RH).
23TABLE 23 Results of Stability Tests on Development Lot Time in
Months Test Spec 0 1 2 3 4 5 6 9 12 15 18 Purity .gtoreq.95%
>99.5%.sup.1 >99.5% >99.5% >99.5% >99.5% >99.5%
>99.5% >99.5% >99.5% >99.5% >99.5% Aggregation
.ltoreq.5% 0.6 2.7 0.3 1.2 0.42 0.1 1.30 0.30 0.14 0.078 0.065 PLA2
(14C) + ANA.sup.2 ANA ANA ANA ANA ANA ANA 57%.sup.3 42% 28% 33%
PLA2 (HPLC) + ANA ANA ANA ANA ANA ANA ANA ANA ANA 57% 57
Fibronectin + na na na + na + + + + + + (Fragment) Isoelectric App.
4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 Focusing 4.7 Free Thiol
.ltoreq.10% .ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1%
.ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1%
.sup.1Limit of quantitation for these assays. .sup.2Means Assay was
not available at that time point. .sup.3Ranges in development of
the assay were 21% to 57% for this lot of rhUG.
[0252]
24TABLE 24 Results of Stability Tests on cGMP Lot at 4.degree. C.
Time in Months Test Spec 0 1 2 3 6 9 12 15 Purity .gtoreq.95% 97.4%
99.4% 98.6% 99.1% >99.5% 99.3% >99% 99.2% Aggregation
.ltoreq.5% 2.2% 1.7% 3.0% 1.2% 1.2% 0.5% 0.5% 0.6% PLA2 (14C) +
ANA.sup.1 ANA ANA 39% 39% 66% 69% 76% PLA2 (HPLC) + ANA ANA ANA ANA
ANA ANA 87% 76% Fibronectin + + na + + + + + + (Fragment)
Isoelectric Focusing App. 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 Free
Thiol .ltoreq.10% .ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1%
.ltoreq.1% .ltoreq.1% .ltoreq.1% .ltoreq.1% .sup.1means Assay was
not available at that time point
[0253]
25TABLE 25 Results of Stability Tests on cGMP Lot at 25.degree. C.
and 60% RH Time in Months Test Spec 1 2 4 7 Purity .gtoreq.95%
99.1% 96.3% >99% 98.9% Aggregation .ltoreq.5% 0.53% 0.25% 0.12%
0.34% PLA2 (14C) + 56% 68% 60% 65% PLA2 (HPLC) + ANA 73% 88% 76%
Fibronectin + + + + + (Fragment) Isoelectric App. 4.7 4.7 4.7 4.7
4.7 Focusing Free Thiol .ltoreq.10% <1% <1% <1% <1%
[0254] As shown, both the development lot and the cGMP lot of rhUG
were stable for more than 18 and 15 months, respectively, since
they were originally produced and vialed. These rhUG preparations
have been tested for a number of physical and chemical
characteristics, as well as for biological activity in two potency
assays. Based on these data, these preparations can be expected to
perform the same in vivo, both with respect to each other and with
respect to their original strength and types of biological
activities as described herein and in application Ser. Nos.
08/864,357; 09/087,210; 09/120,264; 09/549,926; 09/861,688;
PCT/US98/11026; PCT/US99/16312; PCT/US00/09979; and
PCT/US01/12126.
[0255] Accordingly, the present invention provides commercially
viable production processes for rhUG, as well as commercially
viable pharmaceutical compositions and formulations.
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